Mechano-activated control of gene expression

ABSTRACT

Described herein are compositions, methods, and systems for modulating Notch receptor activation. Aspects of the invention relate to synthetic proteins comprising at least a Notch NRR (Negative Regulatory Region)-binding scFV fused to a transmembrane domain. Another aspect of the invention relates to drug-dependent synthetic proteins. Constructs and engineered cells comprising the synthetic proteins are additionally described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 15/994,330 filed May 31, 2018, which claimsbenefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No.62/513,031 filed May 31, 2017 and U.S. Provisional Application No.62/586,451 filed Nov. 15, 2017, the contents of each of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2018, isnamed 701586-089500USPT_SL.txt and is 208,433 bytes in size.

TECHNICAL FIELD

The present invention relates generally to compositions and methods forengineering natural and synthetic Notch signaling.

BACKGROUND OF THE INVENTION

Drug-inducible strategies for regulating protein function and geneactivity have been indispensable tools in biological research, yetmethods for controlling diverse systems remain lacking. The Notchprotein is a transmembrane receptor that acts a mechanical “switch,”translating mechanical cues into gene expression. This mechanosensingactivity is achieved via Notch's force-sensitive Negative RegulatoryRegion (NRR), which contains three LNR domains. In the resting state,the LNR domains adopt an autoinhibitory conformation that stericallyhinders proteolytic cleavage necessary for receptor activation. Upon theapplication of a pulling force, however, these LNR domains aredisplaced, and two concomitant proteolytic cleavages occur that releasethe Notch intracellular domain to transport to the nucleus and regulategene expression.

SUMMARY OF THE INVENTION

As described herein, compositions, methods, and systems using antibodydomains have been developed through which signaling from natural andsynthetic Notch receptors can be regulated or modulated. Thesecompositions, methods, and systems can be used to increase the amount offorce required to activate Notch receptors, or to regulate theiractivity on therapeutic cells. The compositions, methods, and systemsdescribed herein are useful for a variety of cell engineeringapplications, including the creation of engineered cells capable ofsensing certain mechanical features of solid tumors (or biomaterials),as well as for precisely controlling therapeutic and/or engineered cellsexpressing synthetic receptor proteins, such as synthetic Notch receptorproteins.

The compositions, methods, and systems described herein involve, inpart, the use of antibody fragments directed against the NRR region ofthe Notch receptor, a force-sensitive mechanical switch that is rupturedduring receptor activation. Binding of these antibodies stabilizes theNRR and prevents Notch activation. Use of scFvs from these antibodiespermits the generation of inhibitory “modules” which are used togenerate synthetic proteins and receptors for precisely controllingsignaling from Notch and synthetic Notch systems. For example, syntheticnotch receptors have been created that allow for gene expression to becontrolled upon binding of the receptor to various ligands of interest(e.g., surface proteins on cancer cells), such as those described inUS20160264665, the contents of which are herein incorporated byreference in their entireties.

As described herein, antibody fragments from anti-NRR antibodies areused as modules for engineering synthetic proteins and receptors forcell-engineering applications. These antibody-derived fragments are usedas new genetic tools to reprogram natural and synthetic Notch signalingfor synthetic biology applications. In some embodiments, these can beused to allow cells expressing these systems to function as geneticallyencoded “tensometers,” permitting, for example, engineered T cells toactivate their cell killing activity in response to the mechanicalproperties of fibrotic tissues or physical features of solid tumors. Incontrast, previous work involving anti-NRR antibodies relied on the useof purified immunoglobulin as an exogenously applied drug or agent.

The compositions, methods, and systems described herein are alsodirected towards compositions, constructs, and methods for controllingthe binding/activation of Notch receptors through the use of syntheticprotein ligands that incorporate NS3 protease domains as aLigand-Inducible Connection (LInC) from the hepatitis C virus. In theabsence of an NS-inhibitor drug, the ligand domain is cleaved andbecomes incapable of activating the notch receptor. When an NS-inhibitordrug is applied, the NS3 domain remains intact and the ligand is capableof activating the notch receptor. As demonstrated herein, using the NS3cis-protease from hepatitis C virus (HCV) as a LInC or drug-induciblelinker allows precise control of protein function and localization usingclinically tested antiviral protease inhibitors. The versatility of theapproach is demonstrated in the design of drug-sensitive transcriptionfactors (TFs) and transmembrane signaling proteins, as described herein.

Also provided herein, in some aspects, are isolated nucleic acidsequences encoding the synthetic proteins and protein ligands, andengineered cells genetically modified with the nucleic acids encodingthe synthetic proteins and protein ligands described herein.

Accordingly, in some aspects, provided herein are synthetic inhibitorproteins comprising a Notch NRR (Negative Regulatory Region)-bindingscFV fused to a transmembrane domain.

In some aspects, provided herein are synthetic auto-inhibitory proteinscomprising a Notch NRR (Negative Regulatory Region)-binding scFV fusedto a transmembrane domain.

In some embodiments of these aspects and all such aspects describedherein, the Notch NRR comprises a Notch NRR1 of SEQ ID NO: 8.

In some embodiments of these aspects and all such aspects describedherein, the Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8or is a variant of Notch NRR1 of SEQ ID NO: 8.

In other aspects, provided herein are synthetic Notch receptor proteinscomprising, in N-terminal to C-terminal order and in covalent linkage,(i) a ligand binding domain (LBD), (ii) a mutated Notch NRR (NegativeRegulatory Region), (iii) a transmembrane domain, and (iv) anintracellular domain, wherein the mutated Notch NRR is bound withhigh-affinity by a synthetic inhibitor protein comprising a mutatedNotch NRR-binding scFV fused to a transmembrane domain.

In some embodiments of these aspects and all such aspects describedherein, the mutated Notch NRR is mutated relative to Notch NRR1 of SEQID NO: 8.

In some aspects, provided herein are synthetic Notch receptor proteinscomprising, in N-terminal to C-terminal order, and in covalent linkage,(i) a ligand binding domain (LBD), (ii) a scFV that binds to an at leastone Notch NRR (Negative Regulatory Region), (iii) a Notch NRR bound bythe scFV, (iv) a transmembrane domain, and (v) an intracellular domain.

In some embodiments of these aspects and all such aspects describedherein, the Notch NRR comprises a Notch NRR1 of SEQ ID NO: 8.

In some embodiments of these aspects and all such aspects describedherein, the Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8.

In some aspects, provided herein are synthetic, drug-dependent proteincomprising a ligand binding domain (LBD), an NS3 protease domain, and atransmembrane domain.

In some embodiments of these aspects and all such aspects describedherein, the LBD and transmembrane domain comprise a sequence of humanDelta ligand.

In some embodiments of these aspects and all such aspects describedherein, the NS3 domain comprises a sequence of SEQ ID NO: 32.

In some embodiments of these aspects and all such aspects describedherein, the synthetic, drug-dependent protein further comprises atargeting domain.

In some embodiments of these aspects and all such aspects describedherein, the transmembrane domain comprises the human Notch1transmembrane domain of SEQ ID NO: 13 or a variant thereof.

In some embodiments of these aspects and all such aspects describedherein, the scFV comprises, in N-terminal to C-terminal order and incovalent linkage, a VH domain, a linker domain, and a VL domain.

In some embodiments of these aspects and all such aspects describedherein, the scFV is selected from any one of SEQ ID NOs: 15-27.

In some embodiments of these aspects and all such aspects describedherein, the synthetic protein further comprises a signal sequenceN-terminal to the LBD.

Also provided herein, in some aspects, are isolated nucleic acidsequences encoding any of the synthetic proteins described herein.

Also provided herein, in some aspects, are engineered cells comprisingisolated nucleic acid sequences encoding any of the synthetic proteinsdescribed herein.

In some embodiments of these aspects and all such aspects describedherein, the engineered cell is an engineered T cell.

Provided herein, in some aspects, are engineered cells comprising (i) anucleic acid sequence encoding a synthetic inhibitor protein comprisinga Notch NRR (Negative Regulatory Region)-binding scFV fused to atransmembrane domain, and (ii) a nucleic acid sequence encoding asynthetic Notch receptor protein comprising a mutated Notch NRR.

In some embodiments of these aspects and all such aspects describedherein, the engineered cell is an engineered T cell.

In some embodiments of these aspects and all such aspects describedherein, the nucleic acid sequence encoding the synthetic inhibitorprotein, the nucleic acid sequence encoding the synthetic Notch receptorprotein, or both are under operable control of a drug-induciblepromoter.

In some aspects, provided herein, are synthetic, drug-sensitivetranscription factors, comprising: a DNA-binding domain (DB); atranscriptional activation domain (TA); and a HCV NS3 protease domain;wherein the HCV NS3 protease domain is located in between the DB and theTA.

In some embodiments of these aspects and all such aspects describedherein, the HCV NS3 protease domain comprises cleavage activity.

In some embodiments of these aspects and all such aspects describedherein, the cleavage activity activates the transcription factor.

In some embodiments of these aspects and all such aspects describedherein, the DB is Gal4 DB or Cas9 DB.

In some embodiments of these aspects and all such aspects describedherein, the DB is reverse tetracycline repressor.

In some embodiments of these aspects and all such aspects describedherein, the TA is Gal4 TA, VP64 TA, VP64-p65 TA, or VPR TA.

In some embodiments of these aspects and all such aspects describedherein, the synthetic transcription factor further comprises at leastone fluorescent or at least one SNAP tag.

In some embodiments of these aspects and all such aspects describedherein, the tag is located at a N-terminus or a C-terminus of thetranscription factor.

In some embodiments of these aspects and all such aspects describedherein, the transcription factor further comprises at least onetargeting sequence.

In some embodiments of these aspects and all such aspects describedherein, the targeting sequence is a transmembrane domain or a nuclearlocalization sequence.

In some embodiments of these aspects and all such aspects describedherein, the tag is located at a N-terminus or a C-terminus of thetranscription factor.

In some embodiments of these aspects and all such aspects describedherein, the transcription factor further comprises at least one lipidmodification.

In some embodiments of these aspects and all such aspects describedherein, the lipid modification is myristoylation or palmitoylation.

In some embodiments of these aspects and all such aspects describedherein, the at least one lipid modification is located at an N-terminusor a C-terminus of the transcription factor.

Provided herein in some aspects are isolated nucleic acid sequencesencoding any of the synthetic transcription factor described herein.

Provided herein in some aspects are engineered cells comprising theisolated nucleic acid sequences encoding any of the synthetictranscription factor described herein.

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

The term “antibody” broadly refers to any immunoglobulin (Ig) moleculeand immunologically active portions of immunoglobulin molecules (i.e.,molecules that contain an antigen binding site that immunospecificallybind an antigen) comprised of four polypeptide chains, two heavy (H)chains and two light (L) chains, or any functional fragment, mutant,variant, or derivation thereof, which retains the essential epitopebinding features of an Ig molecule. Such mutant, variant, or derivativeantibody formats are known in the art. Nonlimiting embodiments of whichare discussed below, and include but are not limited to a variety offorms, including full length antibodies and antigen-binding portionsthereof; including, for example, an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahuman antibody, a humanized antibody, a single chain antibody, a Fab, aF(ab′), a F(ab′)2, a Fv antibody, fragments produced by a Fab expressionlibrary, a disulfide linked Fv, a scFv, a single domain antibody (dAb),a diabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof, bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference) and/or antigen-binding fragments ofany of the above (See, generally, Hood et al., Immunology, Benjamin,N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual,Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature,323, 15-16 (1986), which are incorporated herein by reference).Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain antigen or target binding sites or “antigen-binding fragments.”The antibody or immunoglobulin molecules described herein can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule, as isunderstood by one of skill in the art. Furthermore, in humans, the lightchain can be a kappa chain or a lambda chain.

In a full-length antibody, each heavy chain is comprised of a heavychain variable domain (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains: CH1, CH2, and CH3. Each light chain is comprised of alight chain variable domain (abbreviated herein LCVR as VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell-known to those skilled in the art. The chains are usually linked toone another via disulfide bonds.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain, and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of anantibody mediates several important effector functions, for example,cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC), and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Antigen-binding functions of an antibody can be performed byfragments of a full-length antibody. Such antibody fragment embodimentsmay also be incorporated in bispecific, dual specific, or multi-specificformats such as a dual variable domain (DVD-Ig) format; specificallybinding to two or more different antigens (e.g., Notch receptor and adifferent antigen molecule). Examples of binding fragments encompassedwithin the term “antigen-binding portion” of an antibody include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL, andCH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al. (1989) Nature, 341: 544-546; PCT PublicationNo. WO 90/05144), which comprises a single variable domain; and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123); Kontermann and Dubeleds., Antibody Engineering, Springer-Verlag, N.Y. (2001), p. 790 (ISBN3-540-41354-5). In addition, single chain antibodies also include“linear antibodies” comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.(1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870).

An immunoglobulin constant (C) domain refers to a heavy (CH) or light(CL) chain constant domain. Murine and human IgG heavy chain and lightchain constant domain amino acid sequences are known in the art.

As used herein, the term “target” refers to a biological molecule (e.g.,peptide, polypeptide, protein, lipid, carbohydrate) to which apolypeptide domain which has a binding site can selectively bind. Thetarget can be, for example, an intracellular target (e.g., anintracellular protein target) or a cell surface target (e.g., a membraneprotein, a receptor protein). Preferably, a target is a cell surfacetarget, such as a cell surface protein.

As described herein, an “antigen” is a molecule that is bound by abinding site on a polypeptide agent, such as a binding protein, anantibody or antibody fragment, or antigen-binding fragment thereof.Typically, antigens are bound by antibody ligands and are capable ofraising an antibody response in vivo. An antigen can be a polypeptide,protein, nucleic acid or other molecule. In the case of conventionalantibodies and fragments thereof, the antibody binding site as definedby the variable loops (L1, L2, L3 and H1, H2, H3) is capable of bindingto the antigen. The term “antigenic determinant” refers to an epitope onthe antigen recognized by an antigen-binding molecule, and moreparticularly, by the antigen-binding site of said molecule.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound by abinding protein. An epitope may be determined by obtaining an X-raycrystal structure of an antibody:antigen complex and determining whichresidues on the antigen are within a specified distance of residues onthe antibody of interest, wherein the specified distance is, 5 Å orless, e.g., 5 Å, 4 Å, 3 Å, 2 Å, 1 Å or any distance in between. In someembodiments, an “epitope” can be formed on a polypeptide both fromcontiguous amino acids, or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or about 8-10 amino acids in a uniquespatial conformation. An “epitope” includes the unit of structureconventionally bound by an immunoglobulin VH/VL pair. Epitopes definethe minimum binding site for an antibody, and thus represent the targetof specificity of an antibody. In the case of a single domain antibody,an epitope represents the unit of structure bound by a variable domainin isolation. The terms “antigenic determinant” and “epitope” can alsobe used interchangeably herein. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. In someembodiments, an epitope comprises of 8 or more contiguous ornon-contiguous amino acid residues in the sequence in which at least50%, 70% or 85% of the residues are within the specified distance of theantibody or binding protein in the X-ray crystal structure.

The terms “specificity” or “specific for” refers to the number ofdifferent types of antigens or antigenic determinants to which a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof as described herein can bind. The specificity of abinding protein, antibody or antibody fragment, or antigen-bindingportion thereof thereof can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation (KD) of an antigen with an antigen-binding protein, is ameasure of the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein, such as a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof: the lesser the value of the KD, the stronger thebinding strength between an antigenic determinant and theantigen-binding molecule. Alternatively, the affinity can also beexpressed as the affinity constant (KA), which is 1/KD). As will beclear to the skilled person, affinity can be determined in a mannerknown per se, depending on the specific antigen of interest.Accordingly, a binding protein, antibody or antibody fragment, orantigen-binding portion thereof thereof as defined herein is said to be“specific for” a first target or antigen compared to a second target orantigen when it binds to the first antigen with an affinity (asdescribed above, and suitably expressed, for example as a KD value) thatis at least 10 times, such as at least 100 times, and preferably atleast 1000 times, and up to 10000 times or more better than the affinitywith which said amino acid sequence or polypeptide binds to anothertarget or polypeptide.

Accordingly, as used herein, “selectively binds” or “specifically binds”or “specific binding” in reference to the interaction of an antibody, orantibody fragment thereof, or a binding protein described herein, meansthat the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope or target) on thechemical species; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody. In certain embodiments, a binding protein or antibodyor antigen-binding fragment thereof that specifically binds to anantigen binds to that antigen with a K_(D) greater than 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, 10⁻¹⁴ M. In otherembodiments, a binding protein or antibody or antigen binding fragmentthereof that specifically binds to an antigen binds to that antigen witha K_(D) between 10⁻⁶ and 10⁻⁷M, 10⁻⁶ and 10⁻⁸ M, 10⁻⁶ and 10⁻⁹M, 10⁻⁶and 10⁻¹⁰ M, 10⁻⁶ and 10⁻¹¹M, 10⁻⁶ and 10⁻¹²M, 10⁻⁶ and 10⁻¹³ M, 10⁻⁶and 10⁻¹⁴M, 10⁻⁹ and 10⁻¹⁰ M, 10⁻⁹ and 10⁻¹¹M, 10⁻⁹ and 10⁻¹²M, 10⁻⁹ and10⁻¹³ M, 10⁻⁹ and 10⁻¹⁴M. In some embodiments, a binding protein orantibody or antigen-binding fragment thereof binds to an epitope, with aK_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or less. Specific binding can be influencedby, for example, the affinity and avidity of the polypeptide agent andthe concentration of polypeptide agent. The person of ordinary skill inthe art can determine appropriate conditions under which the polypeptideagents described herein selectively bind the targets using any suitablemethods, such as titration of a polypeptide agent in a suitable cellbinding assay. In certain embodiments, a binding protein or antibody orantigen-binding fragment thereof is said to “specifically bind” anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules. Binding proteins,antibodies or antigen-binding fragments that bind to the same or similarepitopes will likely cross-compete (one prevents the binding ormodulating effect of the other). Cross-competition, however, can occureven without epitope overlap, e.g., if epitopes are adjacent inthree-dimensional space and/or due to steric hindrance.

The term “antibody fragment,” or “antigen-binding fragment” as usedherein, refer to a protein fragment that comprises only a portion of anintact antibody, generally including an antigen binding site of theintact antibody and thus retaining the ability to bind antigen. Examplesof antibody fragments encompassed by the present definition include: (i)the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab′fragment, which is a Fab fragment having one or more cysteine residuesat the C-terminus of the CH1 domain; (iii) the Fd fragment having VH andCH1 domains; (iv) the Fd′ fragment having VH and CH1 domains and one ormore cysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) whichconsists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)2fragments, a bivalent fragment including two Fab′ fragments linked by adisulphide bridge at the hinge region; (ix) single chain antibodymolecules (e.g., single chain Fv; scFv) (Bird et al., Science242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988));(x) “diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi)“linear antibodies” comprising a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH-VL dimer.Collectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′) 2 antibody fragments comprise a pair of Fabfragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains, which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that canbe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the invention can bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or can be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” can also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) or Marks et al., J. Mol.Biol. 222:581-597 (1991), for example. A monoclonal antibody can be ofany species, including, but not limited to, mouse, rat, goat, rabbit,and human monoclonal antibodies. Various methods for making monoclonalantibodies specific for an antigen, such as Notch, as described herein,are available in the art. For example, the monoclonal antibodies can bemade using the hybridoma method first described by Kohler et al.,Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Pat. No.4,816,567). “Monoclonal antibodies” can also be isolated from orproduced using phage antibody libraries using the techniques originallydescribed in Clackson et al., Nature 352:624-628 (1991), Marks et al.,J. Mol. Biol. 222:581-597 (1991), McCafferty et al., Nature, 348:552-554(1990), Marks et al., Bio/Technology, 10:779-783 (1992)), Waterhouse etal., Nuc. Acids. Res., 21:2265-2266 (1993), and techniques known tothose of ordinary skill in the art.

The term “human antibody” includes antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the disclosure may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody” does not include antibodies in whichCDR sequences derived from the germline of another mammalian species,such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody” includes all human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes, orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable domains linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from one species but in whichthe sequences of one or more of the CDR regions of VH and/or VL arereplaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable domains in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable domains of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable domains. Theterm “CDR set” as used herein refers to a group of three CDRs that occurin a single variable domain capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable domainof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothiaet al. (1989) Nature 342: 877-883) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan et al. ((1995) FASEB J. 9:133-139) and MacCallum etal. ((1996) J. Mol. Biol. 262(5):732-745). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughexemplary embodiments use Kabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. ((1987) J. Mol. Biol. 196: 901-917); andChothia et al. ((1992) J. Mol. Biol. 227: 799-817), both areincorporated herein by reference). According to Chothia et al., criticalportions of the CDRs of many antibodies have nearly identical peptidebackbone confirmations despite great diversity at the level of aminoacid sequence. Each canonical structure specifies primarily a set ofpeptide backbone torsion angles for a contiguous segment of amino acidresidues forming a loop.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; i.e., CDR1,CDR2, and CDR3), and Framework Regions (FRs). Each heavy chain iscomposed of a variable region of the heavy chain (VH refers to thevariable domain of the heavy chain) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (VL refers to the variable domain of the light chain) and aconstant region of the light chain. The light chain constant regionconsists of a CL domain. The VH and VL regions can be further dividedinto hypervariable regions referred to as complementarity-determiningregions (CDRs) and interspersed with conserved regions referred to asframework regions (FR). Each VH and VL region thus consists of threeCDRs and four FRs that are arranged from the N terminus to the Cterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.This structure is well known to those skilled in the art. According tothe methods used herein, the amino acid positions assigned to CDRs andFRs can be defined according to Kabat (Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991)). Amino acid numbering of antibodies or antigen bindingfragments is also according to that of Kabat.

The term “multivalent binding protein” denotes a binding proteincomprising two or more antigen binding sites. A multivalent bindingprotein may be engineered to have three or more antigen binding sites,and is generally not a naturally occurring antibody. The term“multispecific binding protein” refers to a binding protein capable ofbinding two or more related or unrelated targets.

Similarly, unless indicated otherwise, the expression “multivalentantibody” is used throughout this specification to denote an antibodycomprising three or more antigen binding sites. For example, themultivalent antibody is engineered to have the three or more antigenbinding sites and is generally not a native sequence IgM or IgAantibody.

In some embodiments, the binding protein is a single chain dual variabledomain immunoglobulin protein. The terms “single chain dual variabledomain immunoglobulin protein” or “scDVD-Ig protein” or scFvDVD-Igprotein” refer to the antigen binding fragment of a DVD molecule that isanalogous to an antibody single chain Fv fragment. scDVD-Ig proteins aredescribed in U.S. Ser. Nos. 61/746,659; 14/141,498 (US application2014/0243228); and Ser. No. 14/141,500 (US application 2014/0221621),which are incorporated herein by reference in their entireties. In anembodiment, the variable domains of a scDVD-Ig protein are antibodyvariable domains. In an embodiment, the variable domains arenon-immunoglobulin variable domains (e.g., receptor).

In some embodiments, the binding protein is a DVD-Fab. The terms“DVD-Fab” or fDVD-Ig protein” refer to the antigen binding fragment of aDVD-Ig molecule that is analogous to an antibody Fab fragment. fDVD-Igproteins are described in U.S. Ser. Nos. 61/746,663; 14/141,498 (USApplication 2014/0243228); and Ser. No. 14/141,501 (US application US2014/0235476), incorporated herein by reference in their entireties.

In some embodiments, the binding protein is a receptor DVD-Ig protein.The terms “receptor DVD-Ig protein” constructs, or “rDVD-Ig protein”refer to DVD-Ig constructs comprising at least one receptor-like bindingdomain. rDVD-Ig proteins are described in U.S. Ser. Nos. 61/746,616; andSer. No. 14/141,499 (US application 2014/0219913), which areincorporated herein by reference in their entireties.

The term “receptor domain” (RD), or receptor binding domain refers tothe portion of a cell surface receptor, cytoplasmic receptor, nuclearreceptor, or soluble receptor that functions to bind one or morereceptor ligands or signaling molecules (e.g., toxins, hormones,neurotransmitters, cytokines, growth factors, or cell recognitionmolecules).

The terms multi-specific and multivalent IgG-like molecules or “pDVD-Ig”proteins are capable of binding two or more proteins (e.g., antigens).pDVD-Ig proteins are described in U.S. Ser. No. 14/141,502 (USApplication 2014/0213771), incorporated herein by reference in itsentirety. In certain embodiments, pDVD-Ig proteins are disclosed whichare generated by specifically modifying and adapting several concepts.These concepts include but are not limited to: (1) forming Fcheterodimer using CH3 “knobs-into-holes” design, (2) reducing lightchain missing pairing by using CH1/CL cross-over, and (3) pairing twoseparate half IgG molecules at protein production stage using “reductionthen oxidation” approach.

In certain embodiments, a binding protein disclosed herein is a“half-DVD-Ig” comprised of one DVD-Ig heavy chain and one DVD-Ig lightchain. The half-DVD-Ig protein preferably does not promote cross-linkingobserved with naturally occurring antibodies which can result in antigenclustering and undesirable activities. See U.S. Patent Publication No.2012/0201746 which is incorporated by reference herein in its entirety.In some embodiments, the binding protein is a pDVD-Ig protein. In oneembodiment, a pDVD-Ig construct may be created by combining two halvesof different DVD-Ig molecules, or a half DVD-Ig protein and half IgGmolecule.

In some embodiments, the binding protein is an mDVD-Ig protein. As usedherein “monobody DVD-Ig protein” or “mDVD-Ig protein” refers to a classof binding molecules wherein one binding arm has been renderednon-functional. mDVD-Ig proteins are described in U.S. Ser. No.14/141,503 (US Application 2014/0221622) incorporated herein byreference in its entirety.

The Fc regions of the two polypeptide chains that have a formula ofVDH-(X1)n-C-(X2)n may each contain a mutation, wherein the mutations onthe two Fc regions enhance heterodimerization of the two polypeptidechains. In one aspect, knobs-into-holes mutations may be introduced intothese Fc regions to achieve heterodimerization of the Fc regions. SeeAtwell et al. (1997) J. Mol. Biol. 270:26-35.

In some embodiments, the binding protein is a cross-over DVD-Ig protein.As used herein “cross-over DVD-Ig” protein or “coDVD-Ig” protein refersto a DVD-Ig protein wherein the cross-over of variable domains is usedto resolve the issue of affinity loss in the inner antigen-bindingdomains of some DVD-Ig molecules. coDVD-Ig proteins are described inU.S. Ser. No. 14/141,504, incorporated herein by reference in itsentirety.

The term “bispecific antibody”, as used herein, refers to full-lengthantibodies that are generated by quadroma technology (see Milstein etal. (1983) Nature 305: 537-540), by chemical conjugation of twodifferent monoclonal antibodies (see Staerz et al. (1985) Nature 314:628-631), or by knob-into-hole or similar approaches which introducesmutations in the Fc region (see Holliger et al. (1993) Proc. Natl. Acad.Sci. USA 90(14): 6444-6448), resulting in multiple differentimmunoglobulin species of which only one is the functional bispecificantibody. By molecular function, a bispecific antibody binds one antigen(or epitope) on one of its two binding arms (one pair of HC/LC), andbinds a different antigen (or epitope) on its second arm (a differentpair of HC/LC). By this definition, a bispecific antibody has twodistinct antigen binding arms (in both specificity and CDR sequences),and is monovalent for each antigen it binds.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT Publication No. WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In an exemplary embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody providing or nucleic acid sequence encoding at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. In someembodiments, the term “acceptor” refers to the antibody amino acidproviding or nucleic acid sequence encoding the constant region(s). Inyet another embodiment, the term “acceptor” refers to the antibody aminoacid providing or nucleic acid sequence encoding one or more of theframework regions and the constant region(s). In a specific embodiment,the term “acceptor” refers to a human antibody amino acid or nucleicacid sequence that provides or encodes at least 80%, preferably, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al. (2002) Crit. Rev. Immunol.22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol.484:13-30). One of the advantages provided by various embodiments of thepresent disclosure stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities. Moreover, an isolated antibody may be substantially freeof other cellular material and/or chemicals.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH and VL). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “humanized antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. Accordingly, “humanized” antibodiesare a form of a chimeric antibody, that are engineered or designed tocomprise minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipient oracceptor antibody) in which residues from a hypervariable region of therecipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). As used herein, a “composite human antibody” or“deimmunized antibody” are specific types of engineered or humanizedantibodies designed to reduce or eliminate T cell epitopes from thevariable domains.

One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. Also “humanizedantibody” is an antibody or a variant, derivative, analog or fragmentthereof which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 98% or at least 99% identicalto the amino acid sequence of a non-human antibody CDR. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′).sub.2, FabC, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. In an embodiment, a humanized antibody also comprises at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsboth the light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain. A humanized antibody may beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype including without limitation IgG1, IgG2, IgG3,and IgG4. The humanized antibody may comprise sequences from more thanone class or isotype, and particular constant domains may be selected tooptimize desired effector functions using techniques well known in theart.

With respect to constructing DVD-Ig or other binding protein molecules,a “linker” is used to denote a single amino acid or a polypeptide(“linker polypeptide”) comprising two or more amino acid residues joinedby peptide bonds and used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see, e.g., Holligeret al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994)Structure 2: 1121-1123).

A “human antibody,” “non-engineered human antibody,” or “fully humanantibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous mouse immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody can be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes can be recovered from an individual or can have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Exemplary affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen. Avariety of procedures for producing affinity matured antibodies areknown in the art. For example, Marks et al. Bio/Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992). Selective mutation at selectivemutagenesis positions and at contact or hypermutation positions with anactivity enhancing amino acid residue is described in U.S. Pat. No.6,914,128.

A “functional antigen binding site” of an antibody is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same. For multimericantibodies, the number of functional antigen binding sites can beevaluated using ultracentrifugation analysis as described in Example 2of U.S. Patent Application Publication No. 20050186208. According tothis method of analysis, different ratios of target antigen tomultimeric antibody are combined and the average molecular weight of thecomplexes is calculated assuming differing numbers of functional bindingsites. These theoretical values are compared to the actual experimentalvalues obtained in order to evaluate the number of functional bindingsites.

As used herein, a “blocking” or “neutralizing” binding protein,antibody, antibody fragment, antigen-binding fragment or an antibody“antagonist” is one which inhibits or reduces the biological activity ofthe antigen it specifically binds to the antigen. In certainembodiments, blocking or neutralizing antibodies or antagonistantibodies completely inhibit the biological activity of the antigen.The neutralizing binding protein, antibody, antigen-binding fragmentthereof can bind a target, such as Notch, and reduce a biologicalactivity by at least about 20%, 40%, 60%, 80%, 85%, or more. Inhibitionof a Notch biological activity by a neutralizing binding protein,antibody or antigen-binding fragment thereof can be assessed bymeasuring one or more indicators of Notch biological activity well knownin the art.

An antibody having a “biological characteristic” or “functionalcharacteristic” of a designated antibody is one which possesses one ormore of the biological properties of that antibody which distinguish itfrom other antibodies that bind to the same antigen, including, forexample, binding to a particular epitope, an EC50 value, IC50 value orKD values, as defined elsewhere herein.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Inone embodiment, the antibody mutant will have an amino acid sequencehaving at least 75% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof the species-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i.e., amino acid residue from the same group based on commonside-chain properties, see below) with the species-dependent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

As used herein, a “targeting sequence” refers to a polypeptide sequencesufficient to direct the localization of, e.g., a polypetide, to aspecific subcellular localization. By way of example, a “targetingsequence” can direct the polypeptide to the, e.g., a transmembranedomain, or to the nucleus, e.g., a nuclear localization sequence. Atargeting sequence can be added to a biological molecule (e.g., peptide,polypeptide, protein, lipid, carbohydrate) to direct the polypeptideslocalization. A targeting sequence can result in the irreversible orreversible localization of a polypeptide.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). See alsoJonsson U. et al., (1993) Ann. Biol. Clin., 51:19-26; Jonsson U. et al.,(1991) BioTechniques, 11:620-627 (1991); Johnsson U. et al., (1995) J.Mol. Recognit., 8:125-131; and Johnsson U. et al., (1991) Anal.Biochem., 198:268-277.

The term “binding protein conjugate” or “antibody conjugate” refers to abinding protein or antibody or antigen-binding fragment thereof asdescribed herein chemically linked to a second chemical moiety, such asa therapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, anti-cancer therapies as discussed herein, as well aspertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, a binding protein conjugate or antibody conjugate maybe a detectably labeled antibody, which is used as the detectionantibody.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At211,I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein, antibody or antigen-binding protein, or antigen bindingportion thereof, that exists in the form of a crystal. Crystals are oneform of the solid state of matter that is distinct from other forms suchas the amorphous solid state or the liquid crystalline state. Crystalsare composed of regular, repeating, three-dimensional arrays of atoms,ions, molecules (e.g., proteins such as DVD-Igs), or molecularassemblies (e.g., antigen/binding protein complexes).

By “fragment” is meant a portion of a polypeptide, such as a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof, or nucleic acid molecule that contains, preferably, atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of theentire length of the reference nucleic acid molecule or polypeptide. Afragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200,300, 400, 500, 600, or more nucleotides or 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 190, 200 amino acids or more.

By “reduce” or “inhibit” is meant the ability to cause an overalldecrease preferably of 10% or greater, 15% or greater 20% or greater,25% or greater, 30% or greater, 35% or greater, 40% or greater, 45% orgreater, 50% or greater, 55% or greater, 60% or greater, 65% or greater,70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, 95% or greater, 98% or greater, 99% or greater, or complete or100% in a parameter, activity, or condition being measured.

The terms “cell lines,” “host cells,” and “host cells lines” refer tocells that can be genetically engineered to express a nucleic acidsequence encoding any of the synthetic proteins or components thereofdescribed herein. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, and/or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express asynthetic protein of interest.

The term “mammalian host cell” is used to refer to a mammalian cellwhich is capable of being transfected with a nucleic acid sequence andthen of expressing a selected recombinant protein of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present. Suitable mammalian cells for usein the present invention include, but are not limited to Chinese hamsterovary (CHO) cells, baby hamster kidney (BHK) cells, human HeLa cells,monkey COS-1 cell, human embryonic kidney 293 cells, mouse myeloma NSOand human HKB cells (U.S. Pat. No. 6,136,599). The other cell lines arereadily available from the ATCC.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid, such as a nucleicacid sequence encoding a synthetic Notch protein, i.e., a nucleic acidsequence encoding any such recombinant protein of interest. Recombinantcells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and reintroduced into the cell by artificial means. The termalso encompasses cells that contain a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include those obtained by gene replacement,site-specific mutation, using techniques such as Crispr, and relatedtechniques. A “recombinant protein” is one which has been produced by arecombinant cell.

As used herein, the terms “recombinant cell,” “recombinant cell line,”or “modified cell line” refers to a cell line either transiently orstably transformed with one or more nucleic acid constructs, asdescribed herein. Polynucleotides, genetic material, recombinant DNAmolecules, expression vectors, and such, used in the compositions andmethods described herein can be isolated using standard cloning methodssuch as those described by Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichis incorporated herein by reference). Alternatively, the polynucleotidescoding for a recombinant protein product used in the compositions andmethods described herein can be synthesized using standard techniquesthat are well known in the art, such as by synthesis on an automated DNAsynthesizer.

Peptides, polypeptides and proteins that are produced by recombinantanimal cell lines using the cell culture compositions and methodsdescribed herein can be referred to as “recombinant protein ofinterest,” “recombinant peptide,” “recombinant polypeptide,” and“recombinant protein.” The expressed protein(s) can be producedintracellularly or secreted into the culture medium from which it can berecovered and/or collected. Accordingly, the term “recombinant proteinof interest” refers to a protein or fragment thereof expressed from anexogenous nucleic acid sequence introduced into a host cell.

As used herein, the term “transfection” is used to refer to the uptakeof an exogenous nucleic acid by a cell, and a cell has been“transfected” when the exogenous nucleic acid has been introduced insidethe cell membrane. A number of transfection techniques are well known inthe art and are disclosed herein.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection, the transforming nucleic acid can recombine with that ofthe cell by physically integrating into a chromosome of the cell, can bemaintained transiently as an episomal element without being replicated,or can replicate independently as a plasmid. A cell is considered tohave been stably transformed when the transforming nucleic acid isreplicated with the division of the cell.

As used herein an “expression vector” refers to a DNA molecule, or aclone of such a molecule, which has been modified through humanintervention to contain segments of DNA combined and juxtaposed in amanner that would not otherwise exist in nature. DNA constructs can beengineered to include a first DNA segment encoding anacetylation-resistant engineered PDCL3 polypeptide described hereinoperably linked to additional DNA segments encoding a desiredrecombinant protein of interest. In addition, an expression vector cancomprise additional DNA segments, such as promoters, transcriptionterminators, enhancers, and other elements. One or more selectablemarkers can also be included. DNA constructs useful for expressingcloned DNA segments in a variety of prokaryotic and eukaryotic hostcells can be prepared from readily available components or purchasedfrom commercial suppliers.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells can be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used.

As used herein, “cell culture medium” is a media suitable for growth ofanimal cells, such as mammalian cells, in in vitro cell culture. Cellculture media formulations are well known in the art. Typically, cellculture media are comprised of buffers, salts, carbohydrates, aminoacids, vitamins and trace essential elements. “Serum-free” applies to acell culture medium that does not contain animal sera, such as fetalbovine serum. Various tissue culture media, including defined culturemedia, are commercially available.

BRIEF DESCRIPTION OF THE DRAWINGS

This application file contains at least one drawing executed in color.Copies of this patent application publication with color drawings willbe provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1B depict Notch inhibition by cis-interacting proteins. FIG. 1Ashows an illustration of natural cis-inhibition that occurs when Notchand a Notch ligand are expressed on the same cell surface. Interactionof the Notch receptor with its ligand prevents its activation by ligandson adjacent cells. FIG. 1B shows Notch inhibition using a syntheticcis-interacting protein comprising an inhibitory anti-NRR scFv fused toa transmembrane domain. The scFv “clamps” the NRR, preventing itsrupture and thus inhibiting Notch activation.

FIGS. 2A-2C show mutating the affinity of the scFv to control forcesensitivity. FIG. 2A show a figure depicting the crystal structures of“Ab2” (the scFv used in cis-clamp and as LNR4 domain, left) and theNotch1 NRR (right). Wu et al (2010). Availability of crystal structuresallows structure-guided mutations to be made to the scFv that candecrease affinity and tune the stabilizing effects of the scFv. The R99residue was mutated on the heavy chain of Ab2 to either a lysine (slightreduction in affinity) or an alanine (greater reduction in affinity).FIGS. 2B-2C show co-expression of the Notch receptor with the scFv as aseparate transmembrane cis-inhibitor in reporter cells, and culturedthem (FIG. 2B) on surface-adsorbed ligand or (FIG. 2C) withligand-expressing cells. Unaltered Ab2 has inhibitory effects comparableto DAPT (gamma secretase inhibitor), and residue mutations followexpected trends of restoring Notch activity.

FIGS. 3A-3B illustrate an autoinhibitory approach to controllingmechanical sensitivity of Notch. FIG. 3A illustrates a schematic ofNotch activation. In the resting autoinhibited conformation (i), thethree LNR domains (yellow-orange) sterically block the S2 cleavage sitenecessary for Notch activation. Upon application of force (ii), the LNRdomains are displaced, allowing the activating proteolytic cleavages(iii) that release the intracellular domain (ICD) to regulate geneexpression in the nucleus. FIG. 3B shows the protein structure andschematic of the wild type Notch1 NRR (i), which opens in response toabout 5 pN of force. Protein structure of the Notch1 NRR in the presenceof an NRR-binding scFv (magenta), and schematic of the NRR expressedwith this scFv as a contiguous part of its structure (ii). This scFv isdesigned to act as an “LNR4” domain, which should increase the thresholdof force activation to >5 pN.

FIGS. 4A-4C show expression of Notch receptors with and without LNR4domains in HEK 293FT cells. Cells are made to express Notch-basedreceptors that comprise: a WT Notch LBD, an NRR1 Notch core with orwithout LNR4, and a Gal4-VP64 ICD. FIG. 4A shows immunohistochemistry ofligand binding domain (LBD) and ICD showing that cells are able tostably express Notch-based receptors that include an LNR4 domain. LBD(green) stained with soluble ligand Dll4/Fc; ICD (red) stained withαGal4 antibody. FIG. 4B shows the LNR4 domain successfully binds theNRR. In the WT Notch core, immunostaining the NRR with an NRR-bindingantibody revealed co-localization between the LBD and NRR stains.However, the inclusion of an NRR-binding LNR4 domain sterically inhibitsthe ability to stain the NRR; co-localization between LBD and NRRsignals was not observed. LBD (green) stained with soluble ligandDll4/Fc; NRR (red) stained with αNRR antibody. FIG. 4C depicts westernblot assays in transiently transfected cells that show production offull length receptors at expected masses, with inclusion of an LNR4domain showing the expected increase in apparent mass (211 vs 239 kDa).

FIGS. 5A-5B show LNR4 increases force threshold of Notch activation inTension Gauge Tether (TGT) assay. FIG. 5A depicts the use of ruptureabledouble-stranded DNA tethers. Wang et al (2013). One strand of the TGTattaches to a rigid plate through biotin-avidin binding, while the otherpresents a ligand. The TGT ruptures at a defined force, determined bythe orientation of the ligand and the biotin. If the TGT is weaker thanthe force required to activate the Notch receptor, it ruptures beforethe LNR domains can be displaced. If it is stronger, the LNR domains aredisplaced and activation occurs. FIG. 5B shows flow cytometry data ofmCherry reporter activity that shows that the WT NRR (which activates at5 pN) is activated by both 12 pN and 56 pN TGTs. Receptors with an LNR4domain are activated on 56 pN, but not 12 pN TGTs, indicating decreasedmechanical sensitivity.

FIGS. 6A-6C show a schematic depicting drug-dependent Notch activationby the NS-inhibitor BILN-2061 (triangle). FIG. 6A shows that in theabsence of drug, the DLL extracellular domain is cleaved from thetransmembrane domain (TMD); the resulting soluble DLL ligand is not ableto activate Notch receptors. FIG. 6B demonstrates that in the presenceof drug, NS3 cleavage is blocked and the DLL extracellular domainremains bound to its TMD in the signaling-competent state. Subsequentbinding of the tethered DLL to Notch receptors on an adjacent cell leadsto Notch activation and liberation of the Notch intracellular domain(NICD). The liberated NICD is then free to enter the nucleus to mediategene expression. FIG. 6C shows fluorescence imaging ofDLL-NS3-expressing cells and Notch expressing cells (nuclei). Uponactivation, Notch-expressing cells exhibit YFP nuclear fluorescence fromNICD-mediated activation of a chromosomal histone H2B-YFP gene. In theabsence of drug, H2B-YFP is not detected, indicating the absence ofNotch signaling. In the presence of drug, however, H2B-YFP expression isobserved in Notch-expressing cells adjacent to DLL-NS3-expressing cells,indicating that trans-cellular signaling between these cells isdependent on the inhibition of NS3 activity.

FIGS. 7A-7B depict the structure and activation of Notch receptors. FIG.7A shows domain topology of the Notch receptor. FIG. 7B shows that Notchis activated upon the binding and endocytosis of ligands presented byneighboring cells. The force applied to the receptor during ligandendocytosis serves to unfold the NRR and initiate successive proteolyticcleavages at S2 and S3. Activation results in the liberation of theNICD, such that it can be transported into the nucleus to effecttranscriptional changes.

FIGS. 8A-8D illustrate design and expression of an exemplary hN1-scFv.FIG. 8A shows depictions of an exemplary hN1-scFv with the integratedantibody domain. FIG. 8B shows depictions of hN1-scFv with the reportedx-ray structure of the scFv:NRR complex shown in surface rendering. FIG.8C shows the structure of the scFv:NRR complex with the scFv show as aribbon diagram̂LNRs. FIG. 8D shows fluorescence images of live cellsstained with a dye-conjugated version of the Notch ligand DLL4. Membranestaining of hN1-scFv-expressing cells indicate that the chimera iscorrectly trafficked to the surface.

FIGS. 9A-9B show hN1-scFv expression requires increased force foractivation. FIG. 9A shows hN1-scFv-expressing Notch reporter cells thatcontain a NICD-dependent histone-YFP are not activated when co-incubatedcells expressing the Notch ligand DLL1. FIG. 9B shows plating of thesame cells on DLL1-coated coverslips are able to activate hN1-scFv, dueto the ligand ability to apply increased tension to the receptor whenbound to a stiff substrate.

FIG. 10 shows that recombinant hN1 is predominantly localized to the ERin CHO lines. Expression of anti-NRR-scFv-TMD increases the level of hN1localized to the cell surface.

FIGS. 11A-11D show drug dependent localization and activity of dCNV.FIG. 11A is an illustration depicting the self-immolation of dCNV.cis-cleavage leads to the separation of dCas9 from its fused nuclearlocalization (NLS) and transactivation (VPR) domains. FIG. 11B showsthat dCNV is stabilized upon exposure to the NS inhibitor ciluprevir.Immunoblot using anti-Cas9 shows complete cleavage of dCNV in theabsence of drug, producing a 160 kDa band (*) corresponding to thecleaved dCas9. In drug treated cells, full-length (**, 260 kDa) dCNV isobserved. FIG. 11C shows images of immunostained cells showingdrug-dependent localization of dCas9, which localizes to the indrug-treated cells due to preservation of the fused NLS. FIG. 11D showsthat dCNV in combination with an sgRNA targeting the promoter regionCXCR4 is used to upregulate the expression of the chemokine receptor ina drug-dependent manner. Relative expression levels were quantified byflow cytometry using an anti-CXCR4.

FIG. 12 demonstrates inducible inhibition of Notch signaling with aTet-operated scFV. Cells expressing Notch-Gal4 alone (top three peaks),with a constitutive scFv (middle three peaks), or with a Tet-operatedscFv (bottom three peaks) are cultured with sender cells expressing noligand (ctrl) or the ligand Dll1. The constitutively expressed scFvinhibits Notch activation in the presence of ligand, while theTet-operated scFv only inhibits activation in the presence of drug(doxycycline).

FIGS. 13A-13C demonstrate a strategy for designing synthetic NRR-scFvpairs. FIG. 13A shows a figure describing the specificity of scFv'sagainst NRR1 and NRR2 to each NRR and various chimeras of the two. Wu etal (2010). Chimeras that bind neither scFv are promising starting pointsfor choosing a synthetic NRR domain whose scFv may be orthogonal to eachWT NRR. FIG. 13B shows sequence alignments of NRR1 and NRR2. FIG. 13Cshows sequence alignments of scFv's against NRR1 and NRR2. FIGS. 13B and13C are paired with the NRR-scFv crystal structure (FIG. 2A). This isvaluable for understanding the regions of each scFv that bind portionsof each NRR and subsequently designing chimeric scFv's.

FIG. 14 shows an exemplary drug-dependent NS3-Gal4 transcription factor.NS3 fused between the BD and AD of Gal4 yields a functionaltranscription factor. This construct is intact and active in thepresence of an NS3-inhibiting drug, but cleaved and inactive in theabsence of drug.

FIGS. 15A-15I shows drug inducible “turn-on” and “turn-off”transcription factors (TFs). FIG. 15A Schematic depicting a generalexemplary “turn-on” TF design and its stabilization in the presence ofan NS3 inhibitor. FIG. 15B Western blot showing the preservation offull-length DBGal4-NS3-TAGal4 (anti-HA; 60.6 kDa) in cells treated withincreasing concentrations of BILN-2061. TF stabilization was accompaniedby a corresponding increase in the expression of the H2B-Citrinereporter protein (46.5 kDa). DBGal4-NS3-TAGal4 was detected using anHRP-conjugated anti-HA antibody, and signal enhancement was achievedthrough application of an HRP-conjugated secondary antibody. Enhanceddetection showed TF stabilization in cells treated with 0.08 μM ofBILN-2061; intact TF remained undetectable in drug-untreated controls.An additional band corresponding to Cho-K1 mediated degradation of thefusion construct was also detected. FIG. 15C Fluorescence images of aCho-K1 reporter cells (UAS-H2B-Citrine) stably expressingDBGal4-NS3-TAGal4 in the absence and presence of 2.5 μM BILN-2061. FIG.15D Comparison of drug-induced reporter expression levels (as measuredby flow cytometry) of cells expressing “turn-on” TFs containing theindicated TA domains. FIG. 15E Schematic of TMD-NS3-Gal4min cleavage andits drug-activated “turn-off” activity. General design of the “turn-off”TFs containing a generic membrane-localizing element FIG. 15FFluorescence images of HEK 293A cells expressing the dual-taggedBFP-TMD-NS3-Gal4-mCherry. The mCherry-tagged TF unit localized to thenucleus in the absence of drug and was targeted to ER and PM in cellstreated with 3 μM BILN-2061. BFP is N-terminal to the TMD; Gal4 istagged with mCherry. Scale bar is 10 μm. FIG. 15G Schematic ofmyr-palm-NS3-Gal4min cleavage and stabilization. FIG. 15H ImmunostainedHeLa cells showing the sequestration and membrane-targeting ofGal4_(min) in drug-treated cells expressing myr-palm-NS3-Gal4min. FIG.15I Flow cytometry analysis of transiently transfected HEK 293FT cellscoexpressing DBrTetR-NS3-TAVP64-p65 and TMD-NS3-Gal4_(m)i_(n) andcontaining TRE BFP and UAS H2B-Citrine reporter constructs. Treatment ofcells with increasing concentration of BILN-2061 induced concurrent TREBFP activation and UAS H2B-Citrine repression. Analyses were carried outin media containing 100 ng/mL doxycycline to induce DBr_(TetR) bindingto tetO-DNA.

FIGS. 16A-16E show drug-control over endogenous gene expression usingdCas9-NS3-NLS/VPR. FIG. 16A Schematic depiction of the drug-induciblestabilization and nuclear localization of dCas9-NS3-NLS/VPR. FIG. 16BWestern blot showing the preservation of full-length dCas9-NS3-NLS/VPRcopies (250 kDa) in cells treated with 3 μM BILN-2061. An unfused dCas9domain (160 kDa) is produced in the absence of drug. FIG. 16CImmunostained HeLa cells showing the subcellular localization of thedCas9 domain in the presence or absence of 3 μM BILN-2061. Nuclearlocalized dCas9 was observed only in drug treated cells. FIG. 16DSchematic of the time-dependent dye labeling strategy used to analyzecells expressing SNAP-dCas9-NS3-NLS/VPR (top), and correspondingfluorescence images of dye-labeled HeLa cells (bottom). Old proteincopies produced in the absence of drug (red) were confined to thecytoplasm, whereas those that were made in the presence of drug (green)were able to translocate into the nucleus. FIG. 16E Drug-inducedupregulation of CXCR4 expression in HEK 293FT cells coexpressingdCas9-NS3-NLS/VPR and sgRNA targeting the endogenous CXCR4 promoter.Expression of the chemokine receptor was analyzed by staining of cellsanti-CXCR4 antibody followed by quantification via flow cytometry.

FIGS. 17A-17F show drug control over ligand presentation andintercellular Notch signaling. FIG. 17A Schematic of representation ofan exemplary design of Dll1-NS3-mCherry and FIG. 17B shows itsdrug-inducible preservation as a cell-surface ligand. FIG. 17CImmunofluorescence staining of fixed (non-permeabilized) cellsexpressing Dll1-NS3-mCherry. Surface presentation of the Dll1extracellular region was induced by treatment of cells with 1.5 μMBILN-2061. FIG. 17D Schematic depiction of trans-cellular signalingbetween Notch “receiver” cells by Dll1-NS3-mCherry expressing “sender”cells in the absence and presence of drug. FIG. 17E Fluorescence imagesof cocultured sender and receiver cells. Sender cells expressed theDll1-NS3-mCherry ligand (magenta). Receiver cells constitutivelyexpressed human Notch1 and H2B-Cerulean (cyan), and conditionallyexpressed H2B-Citrine upon up Notch activation (yellow). Activatedreceiver cells were observed only in cocultures treated with drug (1.5μM BILN-2061). The interface between sender and receiver cells coloniesis denoted by the white dotted line. FIG. 17F Magnified region from theinset shown in (17E) shown as an overlay with the correspondingtransmitted light image.

FIG. 18 show comparison various commercially available NS3 inhibitors. Aclonal Cho-K1 derived cell line containing a stably integratedGal4-dependent reporter gene (UAS H2B-Citrine) and constitutivelyexpressing DBGal4-NS3-TAGal was tested against various NS3 inhibitors.Cells were treated with drug for ˜24 hours and expression of theH2B-Citrine reporter protein was subsequently quantified by flowcytometry.

FIGS. 19A-19B show characterization of the DBrTetR-NS3-TAVP64-p65“turn-on” TF. FIG. 19A The presence of both doxycycline (to induce rTetRbinding to tetO sequences) and an NS3 inhibitor (BILN-2061) was requiredto activate the expression of an BFP reporter protein under control ofthe TRE promoter. FIG. 19B Comparison of BFP expression (as quantifiedby flow cytometry) in HEK 293FT cells co-transfected with anDBrTetR-NS3-TAVP64-p65-encoding plasmid and reporter TRE-BFP DNA andtreated with the indicated drugs.

FIGS. 20A-20B show drug-induced gene downregulation using “turn-off”TFs. FIG. 20A Western blot analysis of HEK 293FT cells transientlytransfected with DNA encoding TMD-NS3-Gal4min or myr-palm-NS3-Gal4min.Intact proteins were preserved in cells treated with 3 μM BILN-2061. TheHEK 293FT cells contained a stably integrated Gal4 dependent reportergene (UAS H2B-Citrine), the expression of which was detected only indrug untreated cells. The full-length mass of TMD-NS3-Gal4_(min) is 96kDa, and full-length myr-palm-NS3-Gal4min is 63 kDa. A positive controlwas carried out in which cells were transfected with a plasmid encodingthe Gal4 DB fused to VP64 (Gal4-VP64).

FIG. 20B Drug-induced downregulation of reporter expression in HEK 293FTreporter cells (UAS H2B-Citrine) transiently transfected with constructsencoding TMD-NS3-Gal4min or myr-palm-NS3-Gal4_(min) and treated with theindicated concentrations of the NS3 inhibitor grazoprevir. Values aredisplayed as mean±s.d., as determined in triplicate.

FIGS. 21A-21B show drug-induced activation of a fluorescent reportergene using dCas9-NS3-NLS/VPR. Flow cytometry FIG. 21A and representativefluorescence images FIG. 21B of H2B-Citrine expression (green) from areporter construct (UAS H2B-Citrine) as mediated by drug-stabilizeddCas9-NS3-NLS/VPR and a corresponding UAS-targeting sgRNA. Flowcytometry analyses of reporter expression levels were compared toactivation as mediated by “dCNV S139A,” a version of dCas9-NS3-NLS/VPRin which the catalytic serine residue of the NS3 protease was mutated toalanine. The sgRNA construct used (Addgene plasmid #6415) also encoded aconstitutively expressed mCherry (depicted as red in the fluorescenceimages).

FIG. 22 sgRNAs targeting the CXCR4 promoter were co-transfected into HEK293FT alongside DNAs encoding either dCas9-VPR, or dCas9-NS3-NLS/VPR.The extent of CXCR4 upregulation was subsequently quantified by flowcytometry using a fluorescently-labeled anti-CXCR4 antibody. Cellscontaining dCas9-NS3-NLS/VPR were analyzed under drug-treated anduntreated conditions (BILN-2061, 3 μM), and compared to catalyticallyinactive dCas9-NS3-NLS/VPR (NS3 “S139A” mutant), dCas9-VPR containing,and non-transfected HEK 293FT cells (control). Antibody staining of livecells was carried out 24 hours following transfection/drug-treatment.Values are displayed as mean±s.d., as determined in triplicate.

FIGS. 23A-23B show dose-dependent nuclear localization of the dCas9domain in cells expressing dCas9-NS3-NLS/VPR. FIG. 23A Transfected HeLacells were treated with the indicated concentration of BILN-2061 for 24h and subsequently fixed and permeabilized before immunostaining withanti-Cas9 antibody and counterstaining with DAPI. FIG. 23B The extent ofnuclear localization was analyzed through through analysis of the pixelintensities along the red lines indicated in (A) and plotted using theImageJ-based software package Fiji.

FIGS. 24A-24B show inducible gene activation using MCP-NS3-VP64. FIG.24A Schematic of inducible dCas9-mediated transcription via conditionalpreservation of a sgRNA-binding protein (MCP-NS3-VP64). MCP bindshairpin-modified sgRNA and localizes the VP64 TA domain to the DBscaffold only in drug-treated cells. FIG. 24B Drug-induced activation ofa TRE-H2B-Citrine reporter protein (yellow) via expression of dCas9combined with MCP-NS3-VP64 and sgRNA targeting the TRE3G promoter. Cellswere co-transfected with fluorescent marker (mTurqoise-2, rendered inred) to identify positively transfected cells. H2B-Citrine was inducedonly in drug-treated cells.

FIGS. 25A and 25B show images of Dll1-NS3/4A-mCherry mediated cell-cellsignaling captured by epifluorescence imaging under 10× magnification.FIG. 25A Sender cells expressing the Dll1-NS3/4A ligand (magenta) arecultured with receiver cells expressing the Notch transmembranereceptor. Receiver cell population is identified by a constitutivelyexpressed H2B-Cerulean (cyan). Notch activation drives reporter activityof H2B-Citrine (yellow). Cells are cultured together with or without 1.5μM BILN-2061 drug for 72 hours before imaging. FIG. 25B

FIGS. 26A-26C show data depicting the flow cytometry gating proceduresused herein. FIG. 26A Live cells were gated using FSC and SSC asdepicted with the black line. FIG. 26B A positive transfection gate wasmade by gating for the top 1% fluorescing WT cells. FIG. 26C Thepositive transfection gate was then applied to all transfected cellpopulations. Geometric mean of reporter fluorescence was then measuredfrom positively transfected cells. Mean intensities from nuclearH2B-Citrine in individual receiver cells was quantified via analysis ofimages from drug-treated and untreated co-cultures. Expression of theNICD-dependent reporter was compared between receiver cells that were indirect contact with sender cells, as well those that were distant fromsender cells. The displayed values are reported as mean intensities,±s.e.m., with n>100 cells per analyzed group.

FIGS. 27A and 27B show time-dependent analysis of the drug-inducedpreservation of Gal4-based TFs containing NS3 upon treatment of cellswith grazoprevir. Stable cell lines expressing either Gal4DB-NS3-Gal4TAor Gal4DB-NS3-VP64 were treated with 5 μM grazoprevir for the indicatedtimes prior to cell lysis in SDS-PAGE loading buffer and subsequentanalysis by western blot. The drug-induced preservation of intact TFcopies was determined via the detection of bands corresponding to theintact masses of each TF. Western blots showing the preservation offull-length copies of FIG. 27A Gal4DB-NS3-Gal4TA (60.6 kDa), and FIG.27B Gal4DB-NS3-VP64 (57.8 kDa) are displayed. Times refer to the numberof minutes in which cells were exposed to drug prior to lysis. Westerndetection of the TFs was achieved via an HRP-conjugated anti-HA primaryantibody, followed by an HRP-conjugated secondary antibody.

FIGS. 28A and 28B show reversibility of drug-induced “turn-on” TFpreservation. FIG. 28A HEK 293FT cells transfected with DNA encodingrTetR-NS3-VP64-p65 were “pulsed” with drug (5 μM BILN-2061, 24 h) andsubsequently “chased” with drug-free media for the indicated times.Following each chase, cells were lysed in SDS PAGE loading buffer andthe lysates were subsequently analyzed via western. IntactrTetR-NS3-VP64-p65 (93.3 kDa) and the VP64-p65 cleavage product (42.0kDa) were detected via fused HA tag. NT refers to a non-transfectedcontrol. FIG. 28B The reversibility of transcriptional activation byGal4DB-NS3-VP64 was analyzed using a luciferase-based reporter assay.Applying BILN-2061 and grazoprevir as inducers, a pulse-chase analysiswas carried out in which cells were treated drug, then withdrawn fromdrug for chase periods of the indicated times. At the end of the timecourse, the luciferase activity of cells was quantified using aluminescence assay. The data were obtained using Cho-K1 reporter cells(UAS-H2B-Citrine) containing a stably-integrated Gal4DB-NS3-VP64construct. The cells were transfected with a Gal4-dependent luciferasereporter construct (5×GAL4-TATA-luciferase) and treated with 3 μMBILN-2061 or grazoprevir 16 h later. The first chase was initiated atthe 12 h time point after drug addiction. “Last 12” refers to controlcells that were transfected and maintained in drug-free media, andtreated for only the last 12 hours preceding cell lysis. Signal from the“Last 12” samples confirmed that the diminished luciferase activitymeasured in the chased cells was not due to their decreased drugexposure durations. Luminescence values were normalized to signal from aco-transfected NanoLuciferase control construct (pNL1.1.TK[Nluc/TK]).Values are displayed as mean±s.d., as determined in triplicate.

FIGS. 29A and 29B show time-dependent western analysis tracking thedegradation of cleaved Gal4_(min) domains. HEK 293FT cells transfectedwith DNAs encoding either TMD-NS3-Gal4_(min) or TMD-NS3-Gal4_(min)-PESTwere grown without inhibitor until treatment with 3 μM grazoprevir atthe indicated times prior to lysis in SDS PAGE loading buffer andsubsequent analysis by western blot. Detection of the intact and cleavedstates of each protein was achieved via labeling with an anti-Gal4 DBantibody on western blots loaded with lysates from cells expressing FIG.29A TMD-NS3-Gal4_(min), or FIG. 29B TMD-NS3-Gal4_(min)-PEST. Bandscorresponding to the full-length version of each construct (“FullConstruct”) were detected only in lanes loaded with lysates fromdrug-treated cells. The intensity of the “Full Construct” bands grewover time, indicating accumulation of the intact proteins following NS3inhibition. Bands corresponding to cleaved Gal4min and Gal4min-PEST werealso observed (“Cleaved TF”), the intensities of which diminished overtime. The half-life of the Gal4min-PEST was attenuated relative to thatof Gal4_(min). The PEST domain used was derived from the C-terminalregion of mouse ornithine decarboxylase, which has previously been usedto generate a “destabilized” version of GFP with a reduced half-life of2 hours.

FIG. 30 shows results using a novel mechanoreceptor with a fluorescentprotein as its bulky ectodomain. Green fluorescent protein has beenshown to unfold at approximately 100 pN (Dietz 2004), and unfolding ofthis domain would reduce steric hindrance and in turn allow the releaseof an intracellular transcription factor through gamma secretaseprocessing. Cells transiently transfected with DNA encoding thisreceptor display increased activation when plated on wells coated withan antibody that binds it. However, this increase in activation onlyoccurred when the antibodies are tethered, and thus are able to applyforce to the receptors: soluble antibodies at similar concentrations donot increase activation. Additionally, this process is supported to begamma secretase dependent, as addition of a gamma secretase inhibitordiminished cell activation. p=0.000269805 (t-test between coatedanti-myc and noncoated for percent cell activation); p=0.000763826(t-test between coated anti-myc and noncoated for fold above receptor).

FIGS. 31A-31C show reduction of SynNotch leakiness by incorporation ofthe juxtamembrane LWF motif (FIG. 31A) Schematic of the Notch TMD andLWF motif, slightly modified from [1] to highlight the differencebetween the original SynNotch core's C-terminus (blue) and the modifiedSynNotch core's C-terminus (red), where 10 amino acids are added toinclude the LWF motif. (FIGS. 31B and 31C) anti-FITC SynNotch receptorscontaining SN (blue) or SN-LWF (red) as the core domain are expressedand tested in HEK 293FT cells. Cells are either plated on fibronectinalone to test background activation (dashed lines) or on fibronectinwith BSA-FITC to activate the receptors (solid lines). FIG. 31Adiscloses SEQ ID NO: 70. In (FIG. 31B), receptors have the Gal4-VP64ICD, while in (FIG. 31C), receptors have the more potent Gal4-VPR ICD.Values shown are the percentage of cells in the ON state for each case.

FIGS. 32A and 32B show expression of sNRR-containing receptors. (FIG.32A) Immunostaining of non-permeabilized HeLa cells expressingGFP-binding SynNotch receptors that contain either the WT NRR (top) orthe engineered sNRR domain (bottom). Labeling of a myc tag (green) atthe N-terminus of the receptors shows that the sNRR domain does notinhibit the cell's ability to express the receptor at its surface. Next,using a soluble antibody to stain the NRR (magenta), it was found thatECD and NRR stains co-localize for the NRR-based receptor, as expectedsince both protein regions are available at the cell surface. The sNRRdomain, however, does not stain for the NRR. This indicates that theincorporated scFv successfully interacts with the NRR domain asintended. (FIG. 32B) Western blot analysis of the cells shown in (A).For both receptors, an αMyc blot is able to detect the full-length (FL)and S1-processed (N-term) forms of the receptor. S1-processing by furinprotease cleaves the receptor into a noncovalently joined heterodimer, afeature important for surface expression and activity of Notchreceptors.

FIGS. 33A and 33B show increased mechanical strength of sNRR domain inTGT assay. (FIG. 33A) TGTs are double stranded-DNA tethers that ruptureat defined forces. One strand of the TGT attaches to a rigid plate, andthe other presents a ligand for cells to bind. In this case, the ligandused is fluorescein, which interacts with an αFITC SynNotch. If thetension tolerance of the TGT is weaker than that of the NRR, the TGTwill break before the NRR can open. If it is stronger, the NRR domainwill unravel, and Notch activation will occur, as visualized byexpression of an H2B-mCherry reporter gene. (FIG. 33B) Flow cytometrydata and fluorescence imaging of HEK 293FT cells expressing NRR- (left)and sNRR-based (right) SynNotch stimulated with various strengths of TGTligands. NRR-based receptors activate in response to TGTs 12 pN andstronger, as expected. sNRR-based receptors do not activate until 56 pNof tension tolerance is provided. It is worthwhile to re-emphasize thatfor the 12, 56, and 100 pN stimuli above, sNRR recepetors are bindingthe same ligand in each instance, but are able to respond differentbased off the underlying mechanical properties of the ligand.

FIGS. 34A-34E show tunability of sNRR mechanical strength. (FIG. 34A)Crystal structure (PDB 3L95) of the soluble antibody used to design sNRRin complex with its NRR antigen. (FIG. 34B) Flow cytometry data from acollection of mutated sNRR domains stimulated vs TGTs. For eachreceptor, values are normalized to their reporter fluorescence on themaximum strength stimulus (>100 pN). (FIG. 34C) Selected flow cytometrydata from (FIG. 34B) presented in detail. The original sNRR receptor isinsensitive to a 12 pN stimulus, and furthermore can discriminatebetween 43, 54, and >100 pN. Mutating a Tyr residue to a Phe (Y49F)slightly weakens the receptor, inducing a marginal response to 12 pN anddecreased discrimination between 43 pN and above. Further mutating to anAla (Y49A) causes ˜50% activation in response to 12 pN and abolishes theability to discriminate between 43 pN and above. The WT NRR is theweakest, responding identically to all mechanical stimuli. Color legendas in (FIG. 34B). (FIG. 34D) Single-cell traces from timelapse imagingof HeLa cells expressing model sNRR domains. This data further confirmsthe distinct tensile strengths of engineered sNRR domains and revealstheir ability to discriminate forces over time. Values are plottedmean±SD, normalized to each receptor's mean max activation in responseto >100 pN. Color legend as in (FIG. 34B). (FIG. 34E) Stochasticmodeling of sNRR-TGT interactions. A given sNRR-TGT pair is modeled ashaving a relative probability of either the TGT rupturing of thereceptor activating, as determined by the relative mechanical strengthsof the two components. Mechanical strengths are modeled as thermaldissociation rates, and four such strengths are considered for sNRR'sand TGT's each, similar to the data presented in (FIG. 34D). Valuesplotted are the number of receptors that get activated during thestochastic model, normalized as in (FIG. 34D), with 10 runs of the modelplotted per pair.

FIG. 35 show force-based gene circuits: myogenic differentiation. αFITCSynNotch receptors with an ICD that drives expression of MyoD areexpressed in C3H 10T1/2 fibroblasts and stimulated with 12 and 54 pNTGTs. Fibroblast differentiation down a myogenic lineage is identifiedby the presence of myosin heavy chain (green) and multinucleation. Whilecells with NRR-based receptors differentiated in response to bothstimuli, cells with sNRR-based receptors only differentiate on 54 pNTGTs.

FIG. 36 shows screening of NRR-binding scFv's. Various antibodies knownto bind the NRR and inhibit Notch activation are incorporated intoSynNotch receptors and stimulated with 12 and 56 pN TGTs. scFv's WC629and WC75 do not offer detectable mechanical stability, as the receptorsare unable to discriminate between the two stimuli, similar to the WTNRR. scFv E6 offers marginal increased mechanostability, while theoriginal sNRR domain offers the greatest mechanical stability, with 56pN only beginning to stimulate the receptor.

DETAILED DESCRIPTION

As described herein, receptors with increased or decreased forceactivation thresholds are both novel and have wide-ranging applications,including, but not limited to, generation of cells with ability todetect physical features of solid tumors, mechanical properties ofbiomaterials, etc. Furthermore, the cis-clamps described herein permitregulation of engineered cells expressing synthetic Notch receptors or“SynNotch receptors,” such as, for example, therapeutic T cells, and areuseful for reducing the known background activity of these receptors. Inaddition, drug-inducible Notch activation described herein allowstighter control of therapeutic interventions that utilize Notch receptortransduction mechanisms.

A continuing goal of synthetic biology is to be able to program newfunctions into cells in ways that can be precisely manipulated forapplications in medicine and basic research. Toward this end,researchers have recombined modular domains from natural signalingproteins and genetic control elements to produce new biological “parts”for cellular engineering applications.¹ However, a limitation has beenthe relatively small number of protein components that are available fordesigning drug-sensitive systems. In particular, domains that can beused to engineer chemical-control into diverse proteins—in ways thatallow them to be tightly and selectively regulated using orthogonaldrugs—remain lacking.

Accordingly, provided herein, in some aspects, are synthetic Notchreceptors or “SynNotch receptors,” having defined and/or programmableforce-activation thresholds for applications in cell engineering, T cellimmunotherapy, and tissue engineering.

Provided herein, in some aspects, are compositions and methods forregulating SynNotch receptor proteins and reducing their backgroundlevels of activity. For example, in some embodiments, genetic regulationof cis-clamps can be used to “turn off” the cell-killing activity ofimmune cells.

Provided herein, in some aspects, are compositions and methods fordrug-inducible control of Notch and/or synNotch activation. Suchcompositions and methods comprise one or more synthetic proteins, suchas a synthetic, drug-dependent protein or a synthetic inhibitor protein.As used herein, a “synthetic protein” refers to a non-naturallyoccurring protein or polypeptide having a desired function for use inthe compositions and methods described herein. Such synthetic proteinscan comprise one or more domains from or derived from a naturallyoccurring protein in combination with one or more domains from orderived from another naturally occurring protein to create a syntheticprotein having desirable functions that are not found togethernaturally. Such domains include naturally occurring domains, as well asmutated or engineered domains derived from naturally occurring domains,or portions of a naturally occurring domain having a desired activity.For example, in some embodiments, a synthetic protein comprises one ormore NS3 protease domains or one or more Notch Regulatory Region(NRR)-binding domains. Other examples of domains that can be used in thesynthetic proteins described herein include transcriptional activationdomains, transcriptional repressor domains, DNA-binding domains, such aszinc-finger-binding domains, protease domains, and the like. Otherdomains contemplated for use in the synthetic proteins described hereininclude extracellular domains, such as ligand-binding extracellulardomains, transmembrane domains, and intracellular domains, such asintracellular signaling domains. In addition, the nucleic acid sequencesencoding the synthetic proteins described herein can comprise additionalsequence elements such as signal sequences and tag sequences.

Accordingly, provided herein, in some aspects, are “cis-clamps” orsynthetic inhibitor proteins. As used herein, a “synthetic inhibitorprotein” comprises a Notch Regulatory Region (NRR)-binding scFv fused toa transmembrane domain and acts to inhibit Notch receptor or syntheticNotch receptor activity. Non-limiting examples of “synthetic inhibitorproteins” described herein include SEQ ID NOs: 42, 42, and 44 andvariants thereof having similar or enhanced inhibitory activity. Notchis typically activated by ligands expressed on adjacent cells, butinhibited when ligands are expressed on the same cell through amechanism known as “cis-inhibition” (FIG. 1A). This cis-interactionserves to prevent cells from receiving signals from their neighbors, andalso prevents spontaneous “ligand independent” background activation,reducing Notch background activity. As demonstrated herein,membrane-tethered anti-NRR scFvs can be used as genetically encodedNotch inhibitors, or “cis-clamps” (FIG. 1B). These scFvs are derivedfrom antibodies that bind and stabilize the NRR region of Notchreceptors, preventing their activation. As shown herein, these“cis-clamps” or synthetic inhibitor proteins can be used to regulateboth endogenous Notch and synthetic Notch (“SynNotch”) activity in amanner similar to ligand cis-inhibition.

As known by those of skill in the art, “single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of an antibody as asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains, whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York,pp. 269-315 (1994).

In some embodiments of the aspects described herein, cis-clamps orcis-inhibitors are used for cell engineering applications, such as foruse in therapeutic T cells. Notch and/or SynNotch receptors are known toexhibit background “leaky” activation. In engineered cells, thesechimeras are used, in some embodiments, as regulatory elements to limitsignaling from synthetic Notch receptors and, in some embodiments, toreduce off-target T cell killing in the case of engineered SynNotch Tcells.

The cis-clamps described herein are especially useful in situationswhere ligand co-expression is problematic. For example, typicallycis-inhibition of a SynNotch receptor is achieved by co-expressing thereceptor and its target ligand on the same cell. However, in the case ofcell immunotherapy, ligands used to activate SynNotch receptors on Tcells are usually cell surface cancer markers, such that co-expressionof these markers would in principle permit cis-inhibition of SynNotchproteins, as well as causing the therapeutic cells to attack oneanother. Thus, the cis-clamps described herein provide a route throughwhich SynNotch receptors can be regulated without the introduction ofcancer marker/antigens to the engineered cells.

In some embodiments, tuning the affinity of an scFv is performed toengineer mechanical sensitivity, as shown in FIGS. 2A-2C, where anNRR-binding scFv is mutated and expressed as a separate transmembranecis-clamp. Non-limiting examples of NRR-binding scFv sequences that canbe used or be further engineered or modified to be used in someembodiments of the synthetic inhibitor proteins described herein includeSEQ ID NOs: 15-27.

In those embodiments where amino acid sequence modification(s) of anscFv, such as an scFv of any one of SEQ ID NO: 15-27, is performed toengineer mechanical sensitivity, amino acid sequence variants of theNRR-binding scFv are prepared by introducing appropriate nucleotidechanges into the nucleic acid encoding the scFv, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the scFv or antibody from which it is derived.Any combination of deletion, insertion, and substitution is made toarrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes also canalter post-translational processes of the scFv, such as changing thenumber or position of glycosylation sites.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions forantibody-based sequences include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.Other insertional variants of an antibody molecule include the fusion tothe N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis typically are the hypervariable regions ofthe VH and/or VL domains of the scFv.

Substantial modifications in the biological properties of an scFv can beaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids canbe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu(L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar:Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3)acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof antibodies or antibody fragments thereof can be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)can be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody. Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theparent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine can also be used.

Addition of glycosylation sites to the antibodies or antibody fragmentsthereof described herein is accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationcan also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of thescFVs used herein are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibody.

Provided herein, in some aspects, are synthetic Notch receptors withmutated or engineered NRR domains. Mutating the NRR domain and utilizingan scFv that has high affinity to the mutated NRR (but not the nativeNRR) in either the cis-clamp or auto-inhibitory receptor configurationsallow for a more specific system with reduced off-target effects, suchas, e.g., the scFv binding the NRR region on notch receptors of adjacentcells).

Also provided herein, in some aspects, are synthetic auto-inhibitoryNotch receptors that comprise an NRR-binding scFv portion. Similar tothe aspects directed to cis-clamps, the scFv portion in theauto-inhibitory Notch receptor stabilizes the NRR region, resulting inmore force being required to activate the receptor. In some embodiments,NRR-binding scFv portions are expressed as a contiguous part ofNotch-based synthetic receptors to act as a synthetic “fourth LNRdomain” (LNR4), offering additional stability to the NRR and increasingthe force threshold of Notch activation. This exemplary approach isdescribed in FIGS. 3A-3B.

In some embodiments, the LNR4 domain can be mutated to change theaffinity of binding to the NRR in order to tune the mechanicalsensitivity of the receptor, as shown in FIGS. 2A-2C for the cis-clampembodiment.

In some aspects, provided herein are constructs, compositions, andmethods for controlling the binding/activation of endogenous and/orsynthetic Notch receptors through the use of synthetic proteins actingas ligands and comprising viral protease domains, such as NS3 proteasedomains from the hepatitis C virus (HCV). The NS3 domain is a serineprotease embedded within the HCV polyprotein that excises itself fromthe precursor polypeptide by cleaving recognition sites flanking it ateither end. The enzyme has been a prime drug target of thepharmaceutical industry due to its sequence and structural distinctionfrom human proteases; multiple selective inhibitors against the “NS3”cis-protease are currently in use for treating HCV infections worldwide,and several new compounds are currently under evaluation by the FDA.

As demonstrated herein, Notch-signaling can be made drug-dependent byinsertion of a protease domain, such as the NS3 protease domain of SEQID NO: 32 or variants thereof, between the extracellular andtransmembrane domains of the Notch ligand Delta or “DLL” (FIGS. 6A-6C).In the absence of drug, the DLL extracellular domain is cleaved from itsmembrane anchor via NS3, releasing a soluble DLL ligand that is able tobind Notch but cannot activate it. Upon inhibition of NS3, however, DLLextracellular domain remains tethered to the cell surface and thus isable to activate Notch on the surface of opposing cells. This aspect ofthe technologies described herein has a variety of applications inbiology and medicine, and is a powerful tool for developmentalsignaling, cancer biology, T cell engineering, and tissue engineering.In some embodiments, this system is used to achieve drug-control over“synthetic Notch” receptors in which the native Notch ligand-bindingdomain is substituted with an alternative protein (such as, e.g.,anti-GFP scFv) for activation by cells expressing complementary surfaceligands (such as, for example, GFP).

Synthetic proteins (or constructs expressing such synthetic proteins)comprising a receptor ligand (DLL, or any other ligand which binds tothe target receptor), NS3 domain, and a targeting domain (e.g., anantibody specific for a cancer cell antigen) can be used, in someembodiments, for therapeutic applications. Once bound to their target(e.g., a cancer cell), these constructs would only activate receptors onadjacent cells when in the presence of an NS-inhibitor drug. Absent thedrug, the NS3 domain would be cleaved, releasing the ligand andpreventing receptor activation.

In some embodiments, the receptor ligand domain or extracellular domainis the extracellular domain of gamma secretase, nicastrin. Gammasecretase is a membrane protein complex involved in biological functionssuch as Notch and amyloid precursor protein (APP) processing. Itsproteolytic subunit, presenilin, acts by catalyzing the cleavage ofintramembrane alpha helices, and in turn allows the release of both theextracellular domain (important in APP pathology) and the intracellulardomain (important in Notch developmental biology). The gamma secretaseextracellular subunit, nicastrin, has been shown to regulate thisprocess through steric hindrance. Gamma secretase substrates with bulkyextracellular domains are resistant to proteolysis, and the regulatedshedding of this bulky ectodomain is a key pathway in Notch processing.

The drug-controllable ligands can be used, in some embodiments, withvarious combinations of other aspects described herein, including thecis-clamp and autoinhibitory Notch receptors.

Also provided herein in some aspects, are modified HCV NS3 proteasedomains, as versatile protein engineering modules that can be applied toinstall drug-sensitivity into both intracellular and cell-surfaceproteins. In its natural context, NS3 is a serine cis-protease thatexcises itself from the HCV polyprotein by cleaving recognition sitesthat flank it at either end.² Because it is essential for HCVreplication, numerous inhibitors targeting the viral protease have beendeveloped. In previous work, NS3 and its inhibitors have been combinedto create tools for conditionally linking proteins to imaging tags anddegradation sequences.³⁻⁶ Given its successful application in thesenon-natural contexts, it was determined whether the viral protease couldbe used to design synthetic and drug-sensitive proteins to gain controlover complex cellular processes such as transcription and intercellularsignaling.

NS3 was tested as a module for engineering drug-sensitive transcriptionfactors (TFs), using the protease as a Ligand-Inducible Connection(LInC) to control the association between modular DNA-binding (DB) andtranscriptional activation (TA) domains. In some embodiments, NS3 wasinserted in between minimal DB and TA sequences sourced from the yeastTF Gal4, generating Gal4_(DB)-NS3-Gal4_(TA) (FIG. 15A). In thisconfiguration, it was expected that the viral protease would serve as aself-immolating linker, excising itself from the fusion construct and,in doing so, separating the DB and TA elements. However, in the presenceof an NS3 inhibitor, it was believed that self-excision of the proteasewould be blocked, resulting in the preservation of full-length TFcapable of activating the expression of targeted genes.

To determine whether DBGal4-NS3-TAGal4 behaved in this manner, theprotein was stably expressed in a mammalian cell line containing aGal4-dependent fluorescent reporter construct (UAS H2B-Citrine).Immunoblotting confirmed that intact DBGal4-NS3-TAGal4 accumulated incells treated with the selective NS3 inhibitor BILN-2061, whilefull-length TF was not detected in drug-untreated controls (FIG. 15B).In addition to TF stabilization, analyses by fluorescence imaging, flowcytometry, and antibody detection showed that treatment with NS3inhibitors also induced expression of H2B-Citrine in a dose-dependentmanner (FIGS. 15B-15C). Various commercially available NS3 inhibitorswere evaluated and multiple compounds capable of inducing robusttranscriptional responses were identified, including BILN-2061,asunaprevir, danoprevir, and grazoprevir (FIG. 18). It was noted thatthe α-ketoamide-based inhibitors tested (telaprevir and boceprevir) didnot induce detectable levels of reporter expression above background.Together, these results indicate that NS3 inhibitors can be used toprecisely regulate the association of protease-linked modules in orderto achieve inducible control over gene expression.

“NS3 inhibitors,” as used herein, refer to inhibitors of NS3 proteasedomain activity. Typically, NS3 protease inhibitors have been classifiedinto two groups. (1) The first generations inhibitors (boceprevir andtelaprevir) are linear α-ketoamide derivatives. These two inhibitorsform a covalent bond with the active site of the enzyme in a reversibleway. The second generation of inhibitors are mostly linear andmacrocyclic noncovalent inhibitors of the NS3-4A enzyme. Accordingly, insome embodiments, non-limiting examples of NS3 inhibitors that can beused with the synthetic protein compositions and methods describedherein comprising NS3 protease domains, such as SEQ ID NO: 32, orvariants thereof, include paritaprevir, grazoprevir, CH 503034(Boceprevir), VX-950 (Telaprevir), BI 201335, SCH 900518 (Narlaprevir),SCH6 (SCH446211), BILN 2061 (Ciluprevir), TMC435 (Simeprevir),ITMN-191/RG7227 (Danoprevir), MK-7009 (Vaniprevir), GS-9256, ACH-1625,MK-5172, ABT-450, IDX320, BMS-650032 (Asunaprevir), ACH-806 (GS-9132),and PHX1766.

Given the modular framework of the system, it was tested whetherdifferent transcription factors possessing tailored properties could bereadily engineered. For example, it was tested whether substitution ofthe Gal4 TA domain with more potent transcriptional effectors (such asVP64, VP64-p65, and VPR)⁷ would yield TFs with enhanced drugsensitivity. Indeed, DBGal4-NS3-TAVP64-p65 and DBGal4-NS3-TAVPR resultedin higher reporter expression at decreased drug concentrations(including robust expression at drug concentrations in the low nanomolarrange) as compared to the initial DBGal4-NS3-TAGal4 (FIG. 15D).Additionally, TFs with activity against alternative promoters were alsodesigned, including one in which the reverse tetracycline repressor(rTetR)⁸ was used as the DB element (DBrTetR-NS3-TAVP64-p65). Intransfected cells, DBrTetR-NS3-TAVP64-p65 exhibited “AND” gate activity,requiring both the presence of doxycycline (to induce rTetR binding totetO sequences) and an NS3 inhibitor in order to activate transcriptionfrom the tetO-containing TRE promoter (FIG. 19). Notably, the effects ofTF preservation and gene activation were reversed following inhibitorremoval (FIG. 28).

To complement the “turn-on” systems described above, a strategy in whichNS3 inhibitors could be used to “turn-off” gene expression was alsodesigned for use in other aspects. In this approach, the NS3 proteasewas used to conditionally link an intact Gal4 (Gal4min) TF to amembrane-targeting domain with the expectation that protease inhibitorcould be used to precisely regulate the amount of soluble versusmembrane-bound TF. Using a Type-I transmembrane protein as a targetingelement, a fusion construct was generated containing NS3 and Gal4min asa C-terminal cytosolic domain (TMD-NS3-Gal4min) (FIG. 15E).

Fluorescence imaging of cells expressing a dual-tagged version of theprotein (BFP-TMD-NS3-Gal4_(m)i_(n)-mCherry) showed that Gal4_(min) wasreleased from its BFP-fused transmembrane domain in drug-untreatedcells, resulting in a liberated TF unit (tagged with mCherry) thatlocalized predominantly to the nucleus (FIG. 15F). However, in cells inwhich NS3 activity had been inhibited, the TF remained linked to itstargeting element and thus trafficked to endoplasmic reticulum (ER)surface and plasma membrane (PM). A version in which an N-terminalmyristoylation and palmitoylation substrate⁹ was used as the targetingdomain (myr-palm-NS3-Gal4min) exhibited similar behavior, becomingoccluded from the nucleus in drug-treated cells (FIGS. 15G-15H).

Given that TFs must localize to the nucleus in order to bind their DNAtargets, whether TMD-NS3-Gal4min and myr-palm-NS3-Gal4min wouldfacilitate target gene expression in reporter cells that could beinducibly downregulated through NS3 inhibition was tested. Confirmingthese were immunoblotting and flow cytometry analyses showing thatconstructs could be used to achieve drug-inducible suppression of aGal4-dependent fluorescent reporter gene (FIG. 20). Consistent withthese results were analyses showing that exposure to NS3 inhibitors ledto the accumulation of membrane-tethered Gal4min (FIGS. 15F-15G, 20A and20B), as well as the gradual depletion of previously-cleaved TF copies(FIGS. 29A and 29B). Measurements by flow cytometry confirmed that drugtreatment suppressed reporter gene expression in a dose-dependent manner(FIGS. 20A and 20B), and live-cell imaging showed that the effect ofdownregulation could be reversed following inhibitor withdrawal.Together, these results demonstrate that TFs can be conditionally linkedto localization signals through NS3, in turn permitting precise controlover their spatial distributions and activities.

Recognizing that natural gene regulation often involves the synchronizedregulation of multiple genes, the “turn-on” and “turn-off” systems werecombined to create a platform for simultaneously regulating distinctpromoters using drug. In cells that coexpressed both a “turn-on” TF and“turn-off” TF (DBrTetR-NS3-TAVP64-P65 and TMD-NS3-Gal4_(m)i_(n),respectively), coinciding and inverse regulation of TRE- andUAS-controlled reporter genes were observed (FIG. 15I). These resultsdemonstrate NS3 can be used to control multiple TFs in individual cellsto activate concurrent and inverse gene expression changes in responseto NS3 inhibition. Such strategies provide powerful approached forgenerating sophisticated, drug-dependent genetic circuits forprogramming complex behaviors into therapeutic mammalian cells.

In addition to TFs targeting engineered promoters, in some aspects,provided herein are drug-sensitive proteins or “synthetic drug-dependentproteins” that can be used to upregulate gene expression from endogenouspromoter sequences, such as the synthetic drug-dependent protein of SEQID NO: 45 or engineered variants thereof. To achieve such control, asdescribed herein, NS3 was integrated into artificial TFs based on dCas9,a catalytically inactive mutant of the Cas9 nuclease that can serve as aprogrammable DNA-binding domain.¹⁰⁻¹¹ First, a LInC module (e.g., NS3protease domain) was integrated into dCas9-VPR¹² in between the DBscaffold and a C-terminal region containing a nuclear localizationsequence (NLS) and the VPR TA element (dCas9-NS3-NLS/VPR) (FIG. 16A). Inthis configuration, it was tested whether NS3 cleavage would not onlyinactivate the TF, but also prevent cleaved dCas9 from translocatinginto the nucleus (e.g., would be cytoplasmically contained).

Western blotting of cell lysates demonstrated that full-length copies ofdCas9-NS3-NLS/VPR accumulated only in cells cultured in the presence ofdrug (FIG. 16B), and fluorescence imaging of immunostained cellsindicated that the dCas9 domain localized to the nucleus in adrug-dependent manner (FIG. 16C, FIG. 21). In addition, live-celltime-dependent dye labeling experiments carried out using a SNAP-tagfused version of the TF (SNAP-dCas9-NS3-NLS/VPR) showed that onlyprotein copies made in NS3-inhibited cells were transported across thenuclear envelope (FIG. 16D). Given that unfused dCas9 molecules canserve as inhibitors of native expression levels (by binding andoccupying targeted DNA sites)¹², the cytoplasmic containment of cleaveddCas9 could serve to prevent undesired gene repression in drug-untreatedcells, as the unfused domain has been reported to suppress geneexpression in certain cases through binding and occupying targeted DNAsites.

To confirm that dCas9-NS3-NLS/VPR can be used to upregulate geneexpression in a drug-inducible manner, the TF was co-expressed withsgRNA sequences targeting either a fluorescent reporter construct (UASH2B-Citrine), or a chromosomal region upstream of the human geneencoding the chemokine receptor CXCR4.¹³ Flow cytometry analyses showedthat BILN-2061 can be used to induce upregulation of both gene targetsin a dose-dependent manner (FIG. 16E, FIG. 22). In addition, tests usingseparate sgRNAs targeting distinct regions of the CXCR4 promoter showedthat (under saturating drug concentrations) dCas9-NS3-NLS/VPR was ableto upregulate receptor expression to a similar extent as dCas9-VPR (FIG.23). A system in which NS3 was used to regulate TA domain associationwith a hairpin-modified sgRNA¹³ was also developed (FIG. 24). Together,these data indicate that NS3 can also be combined with dCas9 to achievetunable transcription of endogenous human genes, and also suggest thatother sophisticated Cas9-based tools¹⁴ could be designed using a similarapproach.

In addition to TFs, certain transmembrane signaling proteins are alsoknown to possess component-based architecture, including the Notchreceptor, its ligands, and their synthetic derivatives.¹⁵⁻¹⁷ Thus, itwas tested whether NS3 can also be used to regulate intercellularsignaling via drug-dependent Notch/SynNotch activation. Toward this end,an NS3-containing version of the Notch ligand Delta-like 1 (Dll1) wasdesigned by integrating the protease into the extracellular portion ofthe protein (Dll1-NS3), positioning it between the receptor-bindingregion and transmembrane domain (TMD) (FIG. 17A). In this configuration,it was tested whether NS3 self-excision would yield a soluble ligandthat, due to its lack of a membrane tether, would not be presented atcell-surface (FIG. 17B). Indeed, immunostaining of cells stablyexpressing Dll1-NS3 showed that presentation of Dll1-NS3 at the cellsurface was induced upon NS3 inhibition (FIG. 17C).

In the prevailing model of Notch activation, the endocytosis ofmembrane-tethered ligand is thought to deliver a mechanical “pulling”energy that is required to trigger the “on” state of the receptor, inturn inducing the release of its intracellular domain (NICD, atranscriptional effector). Because Notch activation requires theendocytosis of membrane-tethered ligands,¹⁸ it was tested whetherdrug-preserved Dll1-NS3 copies would be able to mediate trans-cellularsignaling. To determine whether protease-containing ligand couldactivate Notch signaling in a drug-dependent manner, “sender” cellsexpressing Dll1-NS3 were combined with Notch1-expressing “receiver”cells in a coculture assay (FIG. 17D). Using receiver cells containing aNICD-dependent fluorescent reporter gene (12×CSL H2B-Citrine)¹⁹, Notchactivation at sender cell-receiver cell interfaces was observed only indrug-treated cocultures (FIGS. 17E-17F, FIG. 25). Thus, in addition toserving as a versatile module for controlling intracellular proteins,these results demonstrate that NS3 can also be applied in luminal andcell-surface contexts to regulate cell-cell recognition events and inturn control intercellular communication.

The applications described herein demonstrate that the HCV NS3 proteaseof SEQ ID NO: 32 is a versatile domain that can be used tostraightforwardly engineer drug-sensitivity into both intracellular andcell-surface proteins. Through the implementation of simple andintuitive protein designs, tightly-regulated chemical control overcomplex cellular phenomena was achieved. One significant advantage ofthe methods described herein is the availability of highly-selective NS3inhibitors, many of which have been tested for clinical use and can beobtained from commercial sources. Thus, in addition to its potentialapplications in basic biology investigations, the methods describedherein can also serve as a powerful strategy for regulating therapeuticcells in vivo using safe and clinically-approved antiviral drugs.

In some embodiments, the HCV NS3 protease domain and correspondingrecognition sites can be substituted with other protease domains andrecognition sites from other viruses including, but not limited to,human immunodeficiency virus and human rhinovirus.

In those embodiments of the synthetic or recombinant proteins describedherein where one or more of the protein domains is mutated or engineeredor modified relative to the endogenous or naturally occurring protein,such as a mutated Notch Negative Regulatory Region (NRR), for suchpurposes as enhancing binding or efficacy, or stability, techniquesknown in the art for identifying mutated proteins or domains having oneor more desired properties can be used. For example, modified or mutateddomains or polypeptides can be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) does not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains.

Naturally occurring residues can be divided into groups based on commonside-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp,Glu; (4) basic: His, Lys, Arg; (5) residues that influence chainorientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class. Preferred conservative substitutions for use in thesynthetic proteins described herein are as follows: Ala into Gly or intoSer; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser;Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn orinto Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys intoArg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe intoMet, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyrinto Trp; and/or Phe into Val, into Ile or into Leu. Whether a change inthe amino acid sequence of a synthetic protein results in a functionalvariant can be readily determined by assessing the desired activity ofthe variant synthetic protein or polypeptide relative to the non-mutatedversion of the synthetic protein.

As known to those of skill in the art, receptors tend to have threeregions or domains: an extracellular domain for binding ligands such asproteins, peptides or small molecules, a transmembrane domain thattraverses the cellular membrane, and an intracellular or cytoplasmicdomain which frequently can participate in some sort of signaltransduction event within a cells, such as phosphorylation. Accordingly,in some embodiments of the aspects described herein, the syntheticproteins can comprise various combinations of extracellular,transmembrane, and intracellular domains derived from naturallyoccurring domains or engineered versions of such domains. Non-limitingexamples of transmembrane domains that can be used with the syntheticproteins described herein include SEQ ID NOs: 13, 14, and 31, andengineered or mutant variants thereof. Non-limiting examples ofintracellular domains that can be used with the synthetic proteinsdescribed herein include the Notch Intracellular Domain (NICD) of SEQ IDNOs: 11, and engineered or mutant variants thereof.

It is also understood that different elements or domains of thesynthetic proteins can be arranged in any manner that is consistent withthe desired functionality. For example, a synthetic, drug-dependentprotein can comprise an extracellular or ligand binding domain (LBD),such as SEQ ID NO: 2 or an engineered variant thereof, an NS3 proteasedomain of SEQ ID NO: 32 or variant thereof, and a transmembrane domainfrom N-terminal to C-terminal, in some embodiments. In some embodiments,additional domains or amino acid sequences can be included C- orN-terminal to the various domains comprising the synthetic proteinsdescribed herein.

In some embodiments of the aspects described herein, a synthetic proteincomprises one or more domains from or derived from a transcriptionalregulator. Transcriptional regulators either activate or represstranscription from cognate promoters. Transcriptional activatorstypically bind nearby to transcriptional promoters and recruit RNApolymerase to directly initiate transcription. Transcriptionalrepressors bind to transcriptional promoters and sterically hindertranscriptional initiation by RNA polymerase. Other transcriptionalregulators serve as either an activator or a repressor depending onwhere it binds and cellular conditions. Accordingly, as used herein, a“transcriptional activation domain” refers to the domain of atranscription factor that interacts with transcriptional controlelements and/or transcriptional regulatory proteins (i.e., transcriptionfactors, RNA polymerases, etc.) to increase and/or activatetranscription of one or more genes. Non-limiting examples oftranscriptional activation domains include: a herpes simplex virus VP16activation domain, VP64 (which is a tetrameric derivative of VP16), HIVTAT, a NF B p65 activation domain, p53 activation domains 1 and 2, aCREB (cAMP response element binding protein) activation domain, an E2Aactivation domain, NFAT (nuclear factor of activated T-cells) activationdomain, yeast GAL4, yeast GCN4, yeast HAP1, MLL, RTG3, GLN3, OAF1, PIP2,PDR1, PDR3, PHO4, LEU3 glucocorticoid receptor transcription activationdomain, B-cell POU homeodomain protein Oct2, plant Ap2, or any othersknown to one or ordinary skill in the art. A transcriptional activationdomain can comprise a wild-type or naturally occurring sequence, or itcan be a modified, mutant, or derivative version of the originaltranscriptional activation domain that has the desired ability toincrease and/or activate transcription of one or more genes. In someembodiments, transcription activation domains for use with the syntheticproteins described herein are selected from Gal4, VP64, VP64-p65, andVPR reverse tetracycline repressor.

In some embodiments of the aspects described herein, a synthetic proteincomprises one or more “DNA-binding domains” (or “DB domains”). Such“DNA-binding domains” refer to sequence-specific DNA binding domainsthat bind a particular DNA sequence element. Accordingly, as usedherein, a “sequence-specific DNA-binding domain” refers to a proteindomain portion that has the ability to selectively bind DNA having aspecific, predetermined sequence. A sequence-specific DNA binding domaincan comprise a wild-type or naturally occurring sequence, or it can be amodified, mutant, or derivative version of the original domain that hasthe desired ability to bind to a desired sequence. In some embodiments,the sequence-specific DNA binding domain is engineered to bind a desiredsequence. Non-limiting examples of proteins having sequence-specific DNAbinding domains that can be used in synthetic proteins described hereininclude GAL4, GCN4, reverse tetracycline receptor, THY1, SYN1, NSE/RU5′,AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, zinc finger proteins ordomains thereof, CRISPR/Cas proteins, such as Cas9, Cas3, Cas4, Cas5,Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2(or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3,Csf4, and Cul96, and TALES.

In those embodiments where a CRISPR/Cas-like protein is used, theCRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, amodified CRISPR/Cas protein, or a fragment of a wild type or modifiedCRISPR/Cas protein. The CRISPR/Cas-like protein can be modified toincrease nucleic acid binding affinity and/or specificity, alter anenzymatic activity, and/or change another property of the protein. Forexample, nuclease (i.e., DNase, RNase) domains of the CRISPR/Cas-likeprotein can be modified, deleted, or inactivated. Alternatively, theCRISPR/Cas-like protein can be truncated to remove domains that are notessential for the functions of the systems described herein. In someembodiments of the engineered systems, methods, and compositions thereofdisclosed herein, a CRISPR enzyme that is used as a DNA binding proteinor domain thereof is mutated with respect to a corresponding wild-typeenzyme such that the mutated CRISPR or domain thereof lacks the abilityto cleave a nucleic acid sequence containing a DNA binding domain targetsite. For example, in some embodiments, a D10A mutation is combined withone or more of H840A, N854A, or N863A mutations to produce a Cas9 enzymesubstantially lacking all DNA cleavage activity.

In some embodiments of the synthetic proteins described herein, one ormore additional “fusion” domains can be added to the synthetic proteinto provide additional desired functionality. Well known examples offusion domains include, but are not limited to, polyhistidine, Glu-Glu,glutathione S transferase (GST), thioredoxin, protein A, protein G, animmunoglobulin heavy chain constant region (Fc), maltose binding protein(MBP), or human serum albumin. A fusion domain can be selected so as toconfer a desired property. For example, some fusion domains areparticularly useful for isolation of the fusion proteins by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt-conjugated resins are used. Many of such matricesare available in “kit” form, such as the Pharmacia GST purificationsystem and the QIAexpress™ system (Qiagen) useful with (HIS6 (SEQ ID NO:69)) fusion partners. As another example, a fusion domain can beselected so as to facilitate detection of the synthetic proteins.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. Non-limiting tag sequences that can be used in the syntheticproteins described herein include SEQ ID NOs: 5-7 and 33. In some cases,the fusion domains have a protease cleavage site, such as for Factor Xaor Thrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. Other types of fusion domainsthat can be selected include multimerizing (e.g., dimerizing,tetramerizing) domains and functional domain.

In some embodiments, a cell is transfected or transformed with a nucleicacid sequence encoding the synthetic protein of interest.

As used herein, the term “transfection” is used to refer to the uptakeof an exogenous nucleic acid by a cell, and a cell has been“transfected” when the exogenous nucleic acid has been introduced insidethe cell membrane. A number of transfection techniques are well known inthe art and are disclosed herein. See, e.g., Graham et al., 1973,Virology 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods inMolecular Biology (Elsevier, 1986); and Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous nucleicacids into suitable host cells.

Suitable techniques of transfection for use with the compositions andmethods described herein include, but are not limited to calciumphosphate-mediated transfection, DEAE-dextran mediated transfection, andelectroporation. Cationic lipid transfection using commerciallyavailable reagents including the Boehringer Mannheim TransfectionReagent (N.fwdarw.1-(2,3-Dioleoyloxy)propyl-N,N,N-trimethylammoniummethylsulfate, Boehringer Mannheim, Indianapolis, Ind.) orLIPOFECTIN or LIPOFECTAMIN or DMRIE reagent (GIBCO-BRL, Gaithersburg,Md.) can also be used.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection, the transforming nucleic acid can recombine with that ofthe cell by physically integrating into a chromosome of the cell, can bemaintained transiently as an episomal element without being replicated,or can replicate independently as a plasmid. A cell is considered tohave been stably transformed when the transforming nucleic acid isreplicated with the division of the cell.

As used herein an “expression vector” refers to a DNA molecule, or aclone of such a molecule, which has been modified through humanintervention to contain segments of DNA combined and juxtaposed in amanner that would not otherwise exist in nature. DNA constructs can beengineered to other domains operably linked to nucleic acid segmentsencoding a desired synthetic or recombinant protein of interest. Inaddition, an expression vector can comprise additional DNA segments,such as promoters, transcription terminators, enhancers, and otherelements. One or more selectable markers can also be included. DNAconstructs useful for expressing cloned DNA segments in a variety ofprokaryotic and eukaryotic host cells can be prepared from readilyavailable components or purchased from commercial suppliers.

Expression vectors can also comprise DNA segments necessary to directthe secretion of a polypeptide or protein of interest. Such DNA segmentscan include at least one secretory signal sequence. Secretory signalsequences, also called leader sequences, prepro sequences and/or presequences, are amino acid sequences that act to direct the secretion ofmature polypeptides or proteins from a cell. Such sequences arecharacterized by a core of hydrophobic amino acids and are typically(but not exclusively) found at the amino termini of newly synthesizedproteins. Very often the secretory peptide is cleaved from the matureprotein during secretion. Such secretory peptides contain processingsites that allow cleavage of the secretory peptide from the matureprotein as it passes through the secretory pathway. A recombinantprotein of interest can contain a secretory signal sequence in itsoriginal amino acid sequence, or can be engineered to become a secretedprotein by inserting an engineered secretory signal sequence into itsoriginal amino acid sequence. The choice of suitable promoters,terminators and secretory signals is well within the level of ordinaryskill in the art. Expression of cloned genes in cultured mammalian cellsand in E. coli, for example, is discussed in detail in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold SpringHarbor, N.Y., 2012; which is incorporated herein by reference in itsentirety). Non-limiting examples of signal sequences for use with thesynthetic proteins described herein include SEQ ID NOs: 1 and 41.

After transfection, the host cell can be maintained either transientlytransformed or stably transformed with said nucleic acid or expressionvector. Introduction of multiple nucleic acids or xpression vectors, andselection of cells containing the multiple nucleic acids or expressionvectors can be done either simultaneously or, more preferably,sequentially. The technique of establishing a cell line stablytransformed with a genetic material or expression vector is well knownin the art (Current Protocols in Molecular Biology). In general, aftertransfection, the growth medium will select for cells containing thenucleic acid construct by, for example, drug selection or deficiency inan essential nutrient, which is complemented by a selectable marker onthe nucleic acid construct or co-transfected with the nucleic acidconstruct. Cultured mammalian cells are generally cultured incommercially available serum-containing or serum-free medium. Selectionof a medium appropriate for the particular host cell used is within thelevel of ordinary skill in the art.

Suitable selectable markers for drug selection used with thecompositions and methods described herein include, but are not limitedto, neomycin (G418), hygromycin, puromycin, zeocin, colchine,methotrexate, and methionine sulfoximine.

A cell to be engineered with synthetic proteins or combinations thereofdescribed herein can be any cell or host cell. As defined herein, a“cell” or “cellular system” is the basic structural and functional unitof all known independently living organisms. It is the smallest unit oflife that is classified as a living thing, and is often called thebuilding block of life. Some organisms, such as most bacteria, areunicellular (consist of a single cell). Other organisms, such as humans,are multicellular. A “natural cell,” as defined herein, refers to anyprokaryotic or eukaryotic cell found naturally. A “prokaryotic cell” cancomprise a cell envelope and a cytoplasmic region that contains the cellgenome (DNA) and ribosomes and various sorts of inclusions.

In some embodiments, the cell is a eukaryotic cell. A eukaryotic cellcomprises membrane-bound compartments in which specific metabolicactivities take place, such as a nucleus. In other embodiments, the cellor cellular system is an artificial or synthetic cell. As definedherein, an “artificial cell” or a “synthetic cell” is a minimal cellformed from artificial parts that can do many things a natural cell cando, such as transcribe and translate proteins and generate ATP.

Once a drug resistant cell population is established, individual clonesmay be selected and screened for high expressing clones. Methods ofestablishing cloned cell line are well known in the art, including, butnot limited to, using a cloning cylinder, or by limiting dilution.Expression of the recombinant protein of interest from each clone can bemeasured by methods such as, but not limited to, immunoassay, enzymaticassay, or chromogenic assay. A cell line stably transformed with a firstnucleic acid construct may be then used as host cell for transfectionwith a second or more nucleic acid constructs, and subjected todifferent drug selections.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells can be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used.

As used herein, “cell culture medium” is a media suitable for growth ofanimal cells, such as mammalian cells, in in vitro cell culture. Cellculture media formulations are well known in the art. Typically, cellculture media are comprised of buffers, salts, carbohydrates, aminoacids, vitamins and trace essential elements. “Serum-free” applies to acell culture medium that does not contain animal sera, such as fetalbovine serum. Various tissue culture media, including defined culturemedia, are commercially available, for example, any one or a combinationof the following cell culture media can be used: RPMI-1640 Medium,RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), MinimumEssential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove'sModified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium,and serum-free media such as EX-CELL™. 300 Series (JRH Biosciences,Lenexa, Kans.), among others. Serum-free versions of such culture mediaare also available. Cell culture media can be supplemented withadditional or increased concentrations of components such as aminoacids, salts, sugars, vitamins, hormones, growth factors, buffers,antibiotics, lipids, trace elements and the like, depending on therequirements of the cells to be cultured and/or the desired cell cultureparameters.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation. Accordingly,the terms “comprising” means “including principally, but not necessarysolely”. Furthermore, variation of the word “comprising”, such as“comprise” and “comprises”, have correspondingly the same meanings. Theterm “consisting essentially of” means “including principally, but notnecessary solely at least one”, and as such, is intended to mean a“selection of one or more, and in any combination”. Stated another way,the term “consisting essentially of” means that an element can be added,subtracted or substituted without materially affecting the novelcharacteristics of the invention. This applies equally to steps within adescribed method as well as compositions and components therein. Inother embodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”). For example, a composition that compriseselements A and B also encompasses a composition consisting of A, B andC.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, publications, and websites identified are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents are based on the information availableto the applicants and do not constitute any admission as to thecorrectness of the dates or contents of these documents.

EXAMPLES Example 1

The Notch protein is a transmembrane receptor that acts a mechanical“switch,” translating mechanical cues into gene expression. Thismechanosensing activity is achieved via Notch's force-sensitive NegativeRegulatory Region, which contains three LNR domains. In the restingstate, the LNR domains adopt an autoinhibitory conformation thatsterically hinders proteolytic cleavage necessary for receptoractivation. Upon the application of a pulling force, however, these LNRdomains are displaced, and two concomitant proteolytic cleavages occurthat release the Notch intracellular domain to transport to the nucleusand regulate gene expression. Synthetic notch receptors have beencreated that allow for gene expression to be controlled upon binding ofthe receptor to various ligands of interest (e.g. surface proteins oncancer cells).

Described herein are strategies through which signaling form natural andsynthetic Notch receptors, as disclosed in US patent Application2016/0264665, which is incorporated herein in its entirety by reference,can be regulated/modulated using antibody domains. These systems areused to increase the amount of force required to activate Notchreceptors, or to regulate their activity on therapeutic cells. Thedescribed tools are useful for a variety of cell engineeringapplications, including the creation of engineered cells capable ofsensing certain mechanical features of solid tumors (or biomaterials),as well as permit the precise control over therapeutic/engineered cellsexpressing synthetic Notch receptor proteins.

These systems involve the use of antibody fragments directed against theNRR region of the Notch receptor, a force-sensitive mechanical switchthat is ruptured during receptor activation. Binding of these antibodiesstabilizes the NRR and prevents Notch activation. Use of scFvs fromthese antibodies will permit the generation of inhibitory “modules”which we will use to generate synthetic proteins and receptors toprecisely control signaling from Notch/SynNotch systems.

Antibody fragments from anti-NRR antibodies are used as new modules forengineering synthetic proteins and receptors for cell-engineeringapplications. All previous work involving anti-NRR antibodies haverelied on the use of purified immunoglobulin as an exogenously applieddrug/agent. In the work described herein, these antibody-derivedfragments are used as new “genetic tools” to reprogram natural andsynthetic Notch signaling for synthetic biology applications. Thisallows cells expressing these systems to function as genetically encoded“tensometers,” permitting, for example, engineered T cells to activatetheir cell killing activity in response to the mechanical properties offibrotic tissues or physical features of solid tumors.

Work described herein is also directed towards constructs and methodsfor controlling the binding/activation of notch receptors through theuse of synthetic ligands that incorporate NS3 protease domains from thehepatitis C virus. In the absence of an NS-inhibitor drug, the liganddomain is cleaved and becomes incapable of activating the notchreceptor. When an NS-inhibitor drug is applied, the NS3 domain remainsintact and the ligand is capable of activating the notch receptor.

Non-limiting applications for the compositions, systems, and methodsthereof described herein include:

-   -   a) SynNotch receptors with defined/programmable force-activation        thresholds for applications in cell engineering, T cell        immunotherapy, and tissue engineering.    -   b) Regulating SynNotch proteins and reducing their background        levels of activity. In principle, genetic regulation of        cis-clamps could be used to “turn off” the cell-killing activity        of immune cells.    -   c) Drug-inducible control of notch/synNotch activation

Advantages of the compositions, systems, and methods thereof describedherein include:

-   -   a) Receptors with increased force activation thresholds are        completely novel; potential applications are wide-ranging,        including generation of cells with ability to detect physical        features of solid tumors, mechanical properties of biomaterials,        etc.    -   b) cis-clamps will permit regulation on engineered cells        expressing SynNotch receptors (including therapeutic T cells)        and can be useful for reducing the known background activity of        these receptors.    -   c) Drug-inducible notch activation that allows tighter control        of therapeutic interventions that utilize notch receptor        transduction mechanisms

Notch Cis-Clamps

Some embodiments of the aspects described herein pertain to “cis-clamps”which are synthetic proteins that comprise an NRR-binding scFv fused toa transmembrane domain. Notch is activated by ligands expressed onadjacent cells, but inhibited when ligands are expressed on the samecell through a mechanism known as “cis-inhibition”, as shown in FIG. 1A.This cis-interaction serves to prevent cells from receiving signals fromtheir neighbors, and also prevents spontaneous “ligand independent”background activation, reducing Notch background activity.

Cis-inhibitors are useful for cell engineering, including, for example,therapeutic T cells, as Notch/SynNotch receptors are known to exhibitbackground “leaky” activation. Membrane-tethered anti-NRR scFvs asgenetically encoded Notch inhibitors, or “cis-clamps” as utilized here(FIG. 1B). These scFvs are derived from antibodies that are known tobind and stabilize the NRR region of Notch receptors, preventing theiractivation. These “clamps” can be used to regulate Notch/SynNotchactivity in a manner similar to ligand cis-inhibition. In engineeredcells, these chimeras can be used as regulatory elements to limitsignaling from synthetic Notch receptors and, for instance, reduceoff-target T cell killing in the case of engineered SynNotch T cells. Asdemonstrated in FIG. 12, these genetically encoded inhibitors can beplaced under the control of a drug-inducible system, allowing temporalcontrol over SynNotch T cell activity.

cis-clamps are especially useful in situations where ligandco-expression is problematic. The most obvious route towardcis-inhibition of a SynNotch receptor is to co-express the receptor andits target ligand on the same cell. However, in the case of cellimmunotherapy, ligands used to activate SynNotch receptors on T cellsare usually cell surface cancer markers. While co-expression of thesemarkers would in principle permit cis-inhibition of SynNotch proteins,they could also cause the therapeutic cells to attack one another. Thus,use of cis-clamps provides a novel route through which SynNotchreceptors can be regulated without the introduction of cancermarker/antigens to the engineered cells.

Without wishing to be bound or limited by theory, the work describedherein indicates that tuning the affinity of the scFv allows for theengineering of mechanical sensitivity. Data presented in FIGS. 2A-2Csupport this and shows that NRR-binding scFv is mutated and expressed asa separate transmembrane cis-clamp. Strategies for tuning scFv affinityinvolve, for example, studying available crystal structures of theNotch1 NRR bound to the scFv (Wu et al, 2010) to make structure-guidedresidue mutations. Amino acid residues on the scFvcomplementarity-determining regions (CDRs) that tightly interact withthe NRR are identifiable targets for engineering, where predictions canbe made to substitute residues that will slightly to substantiallymodify scFv affinity, as shown in FIGS. 2A-2C. Alternatively, instead oftargeted mutations, random mutagenesis of these CDRs through techniquessuch as, for example, error-prone PCR, can also be used to perform thedirected evolution of mechanical sensitivity. Other sites within thescFv provide opportunity for tuning include disulfide bridges,alteration of which has been shown to affect overall scFv stability.

Auto-Inhibitory Notch Receptor

Another aspect of the compositions, systems and methods described hereinis directed towards synthetic auto-inhibitory notch receptors thatcontain an NRR-binding scFv. As in the cis-clamp embodiment, the scFv inthe auto-inhibitory notch receptor stabilizes the NRR region resultingin more force being required to activate the receptor.

NRR-binding scFv's is expressed as a contiguous part of Notch-basedreceptors to act as a synthetic “fourth LNR domain” (LNR4), offeringadditional stability to the NRR and increasing the force threshold ofNotch activation. An example of this approach is described in FIGS. 3Aand 3B.

The LNR4 domain can also be mutated to change the affinity of binding tothe NRR in order to tune the mechanical sensitivity of the receptor, aspreviously demonstrated in FIG. 2A for the cis-clamp embodiment.

Notch Constructs with Synthetic NRR Domains

Another aspect of the compositions, systems and methods described hereinis directed towards synthetic notch receptors with mutated NRR domains.Mutating the NRR domain and utilizing an scFv that has high affinity tothe mutated NRR (but not the native NRR) in either the cis-clamp orauto-inhibitory receptor configurations allows for a more specificsystem with reduced off-target effects (e.g., the scFv binding the NRRregion on notch receptors of adjacent cells).

One strategy for creating these synthetic NRR-scFv pairs is to design achimeric NRR domain that contains components from both the Notch1 andNotch2 NRR domains. Similar to scFv that binds NRR1, scFv's that bindand inhibit NRR2 are generated. It has been demonstrated that thesescFv's are specific to their respective NRR domains and that they areunable to bind chimeric NRR1-NRR2 domains with intermixed components.Seeing as these scFv's are specific to each NRR and insensitive tocertain NRR chimeras, in some embodiments, design of a reverse chimera,for example, an αNRR1-αNRR2 scFv fusion that is specific to an NRRchimera but insensitive to WT NRR1 and NRR2. Such an scFv can bedesigned through structure-guided decisions based off the NRR-scFvcrystal structure and amino acid alignments between the two NRRs andscFvs, or it can be generated through traditional methods of antibodyevolution and purification. A synthetic NRR-scFv pair such as thisallows scFv-based regulation of SynNotch activity that is orthogonal tonative Notch signaling. FIGS. 13A-13C provides more details on thisapproach.

Drug-Controllable Notch Signaling

Yet another aspect of the compositions, systems and methods describedherein is directed towards constructs and methods for controlling thebinding/activation of notch receptors through the use of syntheticligands that incorporate NS3 protease domains from the hepatitis C virus(HCV). The NS3 domain is a serine protease embedded within the HCVpolyprotein that excises itself from the precursor polypeptide bycleaving recognition sites flanking it at either end. The enzyme hasbeen a prime drug target of the pharmaceutical industry due to itssequence and structural distinction from human proteases. Multipleselective inhibitors against the “NS3” cis-protease are currently in usefor treating HCV infections worldwide, and several new compounds arecurrently under evaluation by the FDA.

As described herein, in some embodiments, Notch-signaling can be madedrug-dependent by insertion of the NS3 protease domain between theextracellular and transmembrane domains of the Notch ligand Delta (DLL,FIGS. 6A-6C). In the absence of drug, the DLL extracellular domain iscleaved from its membrane anchor via NS3, releasing a soluble DLL ligandthat is able to bind Notch but cannot activate it. Upon inhibition ofNS3, however, DLL extracellular domain remains tethered to the cellsurface and thus is able to activate Notch on the surface of opposingcells. This technology has a variety of applications in biology andmedicine, including, but not limited tom developmental signaling, cancerbiology, T cell engineering, and tissue engineering. This system isapplied, in some embodiments, to achieve drug-control over “syntheticNotch” receptors in which the native Notch ligand-binding domain issubstituted with an alternative protein (such as anti-GFP scFv) foractivation by cells expressing complementary surface ligands (such asGFP).

In addition, SynNotch activation can alternatively be madedrug-dependent by incorporating the NS3 domain into the receptor ratherthan the ligand. For example, in some embodiments, an NS3 domain couldbe placed between the DNA binding domain (BD) and the activating domain(AD) of a Notch ICD transcription factor (such as Gal4). In the absenceof drug, the ICD would lose its DNA-binding ability and becomenonfunctional, whether or not a ligand on a target cell is bound. In thepresence of drug, the ICD would remain intact and would regulate geneactivity only upon ligand-induced receptor activation. FIG. 14demonstrates feasibility of placing NS3 between the BD and AD of Gal4 tocreate a drug-dependent transcription factor with titratable activation.An NS3 domain can also be incorporated into the ECD of Notch, forexample between the NRR and LBD, likewise creating a functional receptoronly in the presence of an NS3-inhibitor.

Compositions, systems and methods described herein comprising a receptorligand (DLL, or any other ligand which binds to the target receptor),NS3 domain, and a targeting domain (e.g. an antibody specific for acancer cell antigen) can be used for a variety of therapeuticapplications. Once bound to their target (e.g., a cancer cell), theseconstructs serve to activate receptors on adjacent cells only when inthe presence of an NS-inhibitor drug. Absent the drug, the NS3 domainwould be cleaved, releasing the ligand, LBD, or ICD BD and preventinggene regulation through receptor activation. The drug-controllableligands and receptors can be used with various combinations of thepreviously described aspects, including the cis-clamp and autoinhibitoryNotch receptors.

Example 2 Chemical and Physical Control Over Gene Expression inMammalian Cells

Provided herein are compositions, systems and methods for measuring andmanaging biomolecules in living cells. These novel compositions, systemsand methods described herein were developed for regulating geneexpression in mammalian systems using chemical and physical cues. Inaddition to serving as valuable tools for studying numerous aspects ofbiology and disease, these technologies are powerful additions to the“toolkit” for engineering therapeutic cells.

Engineering Cellular Responses to Mechanical Cues.

Cells sense forces via surface receptors that can directly affectchanges in gene expression. Described herein is the use of transmembranesignaling protein Notch as a scaffold to engineer new, syntheticmechanoreceptors that are able to activate gene expression in responseto defined and programmable amounts of applied force. These engineeredreceptors are used to design therapeutic mammalian cells capable ofdetecting various mechanical signals, such as matrix elasticity, or theunique physical features of solid tumors.

Remote Control of Therapeutic Cells Using FDA-Approved Antiviral Drugs.

Tools for regulating the therapeutic properties of engineered cellswhile they are in the body, and strategies to eliminate them frompatients following a successful course of treatment, are necessary toensure that cell-based therapeutics can be administered safely. Thenovel compositions, systems and methods described herein were created tocontrol gene expression and signaling in therapeutic cells usingFDA-approved antiviral small molecules, permitting in vivo control overthese agents using safe, orthogonal, and orally-available drugs.

Engineering Cellular Responses to Mechanical Cues.

Notch proteins regulate a variety of cell fate decisions and areactivated through a mechanism involving mechanical force. In mammals,Notch receptors are single-pass transmembrane proteins containing amembrane-proximal domain known as the negative regulatory region (NRR,as shown in FIG. 7A). Biophysical and cellular studies have identifiedthe NRR as a force-activated mechanical switch that serves to regulatethe localization of the Notch intracellular domain (NICD), atranscriptional effector that is cleaved from the receptor andtransported to the nucleus following activation (FIG. 7B).

In its quiescent state, the NRR adopts an autoinhibited conformation inwhich three LNR (LIN12-Notch repeat) modules sterically block a proteasesite (termed S2), which must be cleaved for receptor activation. Notchrecognizes ligands of the Delta/Serrate/lag2 (DSL) and is activated uponthe binding and endocytosis of ligands expressed by neighboring cells.The force applied to the receptor during ligand endocytosis serves todeliver a mechanical “pulling” energy that is able to displace the LNRmodules and reveal the S2 site for cleavage by activatingmetalloproteases. Cleavage at S2 induces additional proteolysis at anintramembrane site (termed S3), which in turn causes the release of NICDfrom the plasma membrane, as shown in FIG. 7A.

Increasing the Force Threshold for Notch Activation:

Recent studies have suggested that molecular interactions within theNRR—specifically, the interactions made by and between the LNRmodules—define the amount of force needed to activate Notch receptors.Without wishing to be bound or limited by theory, it is hypothesizedthat synthetic Notch proteins with increased activation thresholds canbe created through stabilization of the autoinhibited conformation ofthe NRR.

In the work presented herein, human Notch-1 (hN1), a structurallywell-characterized receptor that is activated by ≥5 picoNewtons ofpulling energy, is used as a scaffold for engineering newmechanoreceptors. To increase hN1's force requirement, single chainvariable fragments (scFvs) derived from anti-hN1 antibodies areintroduced that are known to inhibit receptor activation by binding andstabilizing the NRR. These scFvs are integrated into the hN1 ectodomainat positions such that they are able to bind and stabilize their antigen(FIGS. 8A-8B), and previously reported x-ray structures of scFv:NRRcomplexes (FIG. 8C) are used to guide the selection of these positions.

These scFv-containing chimeras exhibit increased force requirementscompared to their natural counterparts, as activation of these receptorsrequires the rupture of the scFv:NRR interaction in addition to that ofthe LNRs. Work described herein shows that an scFv-fused hN1 (hN1-scFv)is correctly processed in the Golgi apparatus and trafficked to the cellsurface to a similar extent as the wild-type hN1 (FIG. 8D). Furthermore,several lines of evidence indicate that the integrated scFv is bound tothe NRR within the receptor, and results also indicate that this bindinginteraction increases the force resistivity of the receptor.

Genetically Encoded “Tensometers” for Sensing the Mechanics of Materialsand Solid Tumors:

Previous work has indicated that the forces required to rupturescFv:antigen complexes are correlated with their thermal dissociationrates, and that these forces can be predictably modulated via mutation.⁶Thus, it is anticipated that scFv-containing Notch proteins should behighly susceptible to engineering, and that the activation thresholds ofthese receptors can be precisely tuned via design, or directedevolution. Single molecule force spectroscopy measurements using variousimmunoglobulin domains indicate that typical scFv:antigen rupture inresponse to forces ranging from 20 to 200 pN⁷—thus, it is possible tocreate Notch receptors with activation thresholds within this regime.

Reinforced Notch receptors have numerous applications in the study ofcellular mechanosensation, as well as in the engineering of therapeuticmammalian cells. These receptors can be used to program cells to detectand execute specified gene expression programs in response to certainphysical features within in their microenvironments. Indeed, workdescribed herein shows that scFv-bound hN1 receptors are able todiscriminate between ligands bound to stiff versus soft substrates,becoming activated only by ligands capable of applying sufficienttension to unbind the scFv from its fused antigen (FIGS. 9A and 9B).These findings show that scFv-containing Notch receptors can act asgenetically encoded “tensometers,” permitting for example, engineered Tcells to activate their cell killing activity in response to themechanical properties of fibrotic tissues or physical features of solidtumors.

Furthermore, cells expressing a NRR-binding scFv exhibit distinctresponses to magnetic fields (compared to hN1-expressing cells) whencultured in the presence of ligand-coated magnetic beads, raising theintriguing possibility that mammalian cells can be engineered to respondto different levels of magnetic force. To define these forces, singlemolecule measurements (using optical and magnetic tweezers) andbiophysical assays (using DNA tension gauge tethers and reporter cells)are used to characterize the mechanics of individual receptors, andmolecular dynamics are used to map the energy landscape of thesereceptors in response to varying degrees of applied energy.

Studies on Notch Receptor Trafficking and Localization:

Recombinant and endogenous hN1 is localized predominantly to the ER in avariety of mammalian cell lines. Expression of a membrane-tetheredNRR-binding scFv (scFv-TMD) dramatically increases the surfacelocalization of recombinant hN1 in CHO cells (FIG. 10), as well as thelocalization of endogenous hN1 in MCF-7 cells.

Furthermore, work described herein shows that low-affinity scFvs areable to increase the surface localization of hN1 without affecting itsability to respond to ligand-expressing cells. “Synthetic Notch”(syn-Notch) receptors (in which the native hN1 ligand-binding region isreplaced with domains recognizing alternative targets, such as cancermarkers), are used to engineer T cells for immunotherapy applications,thus, the described observations may have important implications fortherapeutic cell engineering, as control over receptor localization andconcentration are powerful strategies to regulate the activity of suchsystems.

Remote Control of Therapeutic Cells Using FDA-Approved Antiviral Drugs.

A major challenge associated with the development of cell-basedtherapies is the risk of unintended toxicities stemming from theresidence of engineered cells within patients beyond the course oftreatment. Thus, strategies for eliminating such cells—by inducing theexpression of a “self-destruct” gene after successful treatment, forexample—are necessary to ensure that cell-based therapeutics can beadministered safely.

In recent work, researchers have begun to apply existing drug-induciblegene expression tools as regulatory systems for controlling therapeuticcells—however, many of these systems possess features that complicatetheir translation to clinical applications. For example, rapamycin andtamoxifen are widely used to control the activity of proteins fused todomains recognizing these drugs, but they are also ligands that bind andstimulate endogenous signaling proteins involved in regulatingmetabolism and immunity. Platforms based on the bacterially derivedtetracycline-binding repressor proteins are similarly challenged by themitotoxic properties of tetracyclines as well as their potent antibioticactivity. While the recently described inducible systems based onabscisic acid- and gibberellin-binding proteins have proven to be highlyversatile, use in patients could be complicated by the abundance ofthese plant-derived hormones in the environment and in plant-basedfoods.

Inducible Gene-Expression Using Antiviral Drugs:

To circumvent the challenges imposed by the intrinsic bioactivity ofthese compounds, viral proteases and their corresponding anti-viralinhibitors are used herein to create new inducible systems that areorthogonal to mammalian systems. For example, the activity of diverseproteins can be made drug-sensitive by fusion with the hepatitis C virus(HCV) NS3 protease domain. NS3 is a serine cis-protease that excisesitself from the HCV polyprotein by cleaving recognition sites flankingit at either end. Because it is essential for HCV replication, the NS3protease has been a major target in the drug industry's development ofanti-HCV therapeutics—thus, numerous inhibitors targeting this viraldomain have previously been identified, including several that areFDA-approved.

NS3 protease domain can be used to render transcription factors (TFs)based on the nuclease-deficient dCas9 subject to drug-control. In aninitial design strategy, NS3 is intergrated into the engineered TFdCas9-VPR, (generating dCas9-NS3-VPR, or “dCNV”) placing enzyme betweenthe dCas9 scaffold and the “VPR” transactivation domain (TAD). In thisconfiguration, the protease domain serves as a self-immolating linkerthat leads to the dismemberment of the artificial TF, separating dCas9from its fused nuclear localization signal (NLS) and VPR motif. However,upon exposure to an NS3 inhibitor, self-excision of the protease isblocked and intact copies of the TF are in turn able to transport intothe nucleus to activate the expression of targeted genes. Using an sgRNAsequence complementary to the promoter region of chromosomal DNAencoding the chemokine receptor CXCR4, dCNV was able to activateupregulate expression of the cell surface protein in a drug-dependentmanner.

Drug-Inducible Display of Notch and Syn-Notch Ligands:

NS3 is also used to regulate the presentation of Notch and syn-Notchligands on the surface of engineered mammalian cells. NS3 is integratedinto cell-surface ligands such that they are retained on membranesurfaces (and thus able to activate Notch receptors) only in thepresence of drug. Work described herein demonstrates that drug-dependentNotch signaling by cells expressing and NS3-fused version of the theNotch ligand DLL1 (DLL1-NS3).

Regulation of Therapeutic Cell Activity:

In addition to the further development of new drug-sensitive TFs andsignaling proteins, dCNV and NS3-containing Notch/syn-Notch ligands areapplied to regulate the therapeutic properties of engineered T cells,such as such as target-specificity, cell-killing ability, andself-destruction. A key aspect of the proposed approach is that theseveral FDA approved NS3 inhibitors are already used in the clinic totreat HCV. Thus, these systems are used to modulate therapeutic cells invivo using molecules that are orally available and already known to besafe.

Materials and Methods

DNA Constructs

Plasmid DNA and detailed sequence information for expression vectorsencoding Gal4DB-NS3-Gal4TA, Gal4DB-NS3-VP64, Gal4DB-NS3-VP64-p65,rTetR-NS3-VP64-p65, myr-palm-NS3-Gal4min, dCas9-NS3-NLS/VPR, andSNAP-dCas9-NS3-NLS/VPR can be obtained via AddGene. Standard cloningprocedures were used in the generation of all DNA constructs. ThepEV-UAS-H2B-citrine reporter plasmid was a gift from Michael Elowitz(Caltech), the TRE-mTagBFP reporter plasmid was a gift from Wilson Wong(Boston University), the 5×GAL4-TATA-luciferase reporter plasmid(Addgene #46756) was a gift from Richard Maurer (Oregon Health SciencesUniversity), the Tet-inducible mCherry reporter (Addgene #64128) andsgRNAl_Tet-inducible Luciferase plasmids (Addgene #64128) were giftsfrom Moritoshi Sato (University of Tokyo).

NS3 Inhibitors

Asunaprevir, boceprevir, danoprevir, MK-5172 (a.k.a., grazoprevir), andsimeprevir were from MedChemExpress. Telaprevir was from SelleckChemicals. BILN-2061 was a gift from Roger Tsien and Stephen Adams (UCSan Diego). Concentrated NS3 inhibitor stocks were dissolved in DMSO atconcentrations between 3-10 mM and diluted into cell culture media atthe indicated working concentrations.

Mammalian Cell Culture

All mammalian cell lines were cultured in a humidified incubatormaintained at 37° C. with 5% CO2. HEK 293FT cells (ThermoFisher) werecultured in Dulbecco's modified Eagle medium (DMEM) containing with 10%FBS and supplemented with nonessential amino acids (Life Technologies),Glutamax (Life Technologies), and G418 (500 μg/mL, Invivogen). HeLacells were obtained from ATCC and were cultured in DMEM containing 10%FBS and supplemented with Glutamax and penicillin/streptomycin. Stablecell lines based on CHO T-REx (ThermoFisher) were were maintained inDMEM containing 10% FBS and supplemented with nonessential amino acidsand glutamax.

DNA Transfections

DNA transfections were performed using Lipofectamine 3000 Reagent(ThermoFisher) according to manufacturer's instructions. For imagingexperiments, cells were seeded in dishes or well plates containingcoverslip bottoms either coated with bovine plasma fibronectin (Product#F1141, Sigma-Aldrich) or treated for cell-adherence by the manufacturer(poly-D-lysine by MatTek, or ibiTreat by Ibidi).

Stable Cell Line Generation

Stable cell lines were generated from previously reported CHO-K1 T-RExcells containing stably integrated Gal4- and Notch-dependent reporterconstructs (UAS H2B-Citrine and 12×CSL H2B-Citrine, respectively), whichwere gifts from Michael Elowitz (Caltech). Briefly, cells weretransfected with linearized DNAs encoding the engineered protein ofinterest as well as antibiotic resistance gene for mammalian selection.Transfections were performed in 24-well plates containing 160,000 cellsper well seeded the approximately 24 hours prior to transfection. At 48hours post-transfection, the cells were transferred to 6-well plates andexposed to antibiotic selection using hygromycin (500 μg/mL). Uponelimination of non-transfected control cells (typically after 10 days ofculture in the presence of antibiotic), surviving cells were transferredinto 96-well plates using a limited dilution procedure in order toisolate single clones.

Antibodies

The following primary antibodies were used: mouse anti-Cas9 (Santa CruzBiotechnology, sc-517386, 1:500 dilution for western blotting, 1:50 forimmunostaining), mouse anti-human CD184 (CXCR-4) APC conjugate(BioLegend, 306510, 1:200 dilution for flow cytometry), polyclonal sheepanti-mouse/rat Dll1 (R&D Systems, AF3970, 1:50 dilution forimmunostaining), rabbit anti-Histone H2B (Cell Signalling, 12364,1:1,000 dilution for western blotting), mouse anti-HA-HRP (Santa-Cruz,sc-7392, 1:1,000 dilution for blotting), rabbit anti-GAPDH(Sigma-Aldrich, G9545, 1:3,000 dilution for western blotting), andpolyclonal rabbit anti-Gal4 (Santa Cruz Biotechnology, sc-577, diluted1:500 for western blotting, 1:200 for immunostaining). The followingsecondary antibodies were used: goat anti-human AlexaFluor647 conjugate(ThermoFisher, A-21445, 1:1,000 dilution), donkey anti-sheepAlexaFluor647 conjugate (ThermoFisher, A-21448, 1:1,000 dilution), goatanti-rabbit CF647 conjugate (Sigma-Aldrich, SAB4600184, 1:300 dilution),anti-mouse HRP conjugate (Cell Signalling, 7076, 1:3,000 dilution), andanti-rabbit HRP conjugate (Bio-Rad, 170-6515, 1:3,000 dilution).

Preparation of Cell Lysates for Immunoblotting

Cell lysates used in immunoblotting analyses were prepared by directlysis of drug-treated and untreated cells in 1×LDS-PAGE loading buffer(ThermoFisher) following removal of cell culture media. Such procedurewas applied in order to immediately denature proteins upon lysis, thuspreventing undesired NS3 cis-cleavage in cell lysates. Viscous solutionswere formed upon addition of the lysis reagent, which were clarifiedthrough sonication followed by centrifugation. The lysates weresubsequently analyzed by standard immunoblotting procedures and probedusing the antibodies listed above at the indicated dilutions. Detectionof the labeled antigens was carried by chemiluminescence via theSuperSignal West Pico PLUS Chemiluminescent Substrate (Pierce).

Immunofluorescence Staining of Fixed Cells

Cells were rinsed with PBS prior to fixation with formaldehyde (4% v/v,diluted into PBS from fresh vials containing 16% solutions purchasedfrom ThermoFisher). Cells were fixed for 10 minutes at room temperature,followed by rinsing with PBS (three times) to remove residual fixative.When necessary, cells were then permeabilized with Triton-X 100 (0.2%,v/v, in PBS) for 10 minutes, following by rinsing with PBS. Cells wereblocked with BSA solution (5%, v/v in PBS) for approximately 30 minutesat room temperature prior to staining with primary antibody solution(typically in PBS, or in the appropriate solution as suggested by theantibody supplier) at the dilutions indicated above for 1 hour at roomtemperature. Cells were stained with secondary antibody solution (in PBSat the solutions indicated above) for 1 hour at room temperature beforeimaging.

Time-Dependent Dye Labeling of SNAP-Tagged Proteins

HeLa cells were transfected with DNA encoding SNAP-dCas9-NS3-NLS/VPR asdescribed above herein. Approximately 24 hours later, cells were labeledwith the red fluorescent dye SNAP-Cell TMR STAR (New England Biolabs) incomplete culture media according to the manufacturer's protocol. The dyewas removed by gentle aspiration of the media, followed by rinsing withpre-warmed complete media (three times) to remove residual dye. Cellswere subsequently incubated in culture media containing 3 μM BILN-2061.After 8 hours, cells were then labeled with the green fluorescentSNAP-Cell Fluorescein (New England Biolabs). Cells were fixed with 4%formaldehyde prior to imaging.

Luciferase Assay

CHO-K1 cells stably expressing Gal4DB-NS3-VP64 were transfected with DNAencoding a UAS regulated firefly luciferase reporter construct(5×GAL4-TATA-luciferase). A constitutively transcribed NanoLuciferaseconstruct (pNL1.1.TK[Nluc/TK], Promega) was used as a co-transfectioncontrol. Approximately 16 hours after transfection, cells were treatedwith either BILN-2061, or Grazoprevir (both at 3 μM). Following a 12hour period of drug treatment, a time-series was initiated during whichthe drug-containing media was removed from individual wells and replacedwith drug-free media over the course of a 48 hour period. At the end ofthe series (approximately 56 hours after the initial drug exposure, and72 hours after transfection), the amount of luciferase andNanoLuciferase present in cells was quantified using the Nano-Glo DualLuciferase Reporter Assay System (Promega) according to themanufacturer's protocol. The CHO-K1 cell line used in these analysesalso contained a stably integrated UAS H2B-Citrine reporter construct,and fluorescence imaging confirmed the activation of the Gal4-dependentH2B-citrine gene in all drug treated-wells.

Image Acquisition and Analysis

Cells were imaged by epifluorescence microscopy in imaging-compatiblevessels containing glass coverslip bottoms (MatTek) or optically clearplastic bottoms (ibidi). During imaging, cells were maintained in PBS,standard culture media, or FluoroBrite DMEM (ThermoFisher). Fortime-lapse analyses, cells were imaged in culture media supplementedwith 30 mM HEPES diluted from a 1 M stock (pH 7.2-7.5, ThermoFisher) andmaintained at 37° C. in a heated imaging chamber throughout the durationof the analysis (typically 24 hours). Images were acquired using the ZENimaging software (Zeiss). Image files were processed using theImageJ-based image analysis package Fiji. The images were contrasteduniformly across experiments, and where applicable, pixel intensityprofiles were plotted using the plot profile tool in Fiji. For thetime-lapse analyses, movies were created using the ZEN imaging software.

Flow Cytometry

Cells analyzed by flow cytometry were gated for living cells by scatterdetection. The geometric mean measured reporter fluorescence levels werereported in arbitrary fluorescence units (AFU). Reporter activationanalyses were performed using stable single-clones, or cells transientlytransfected with DNA encoding the analyzed TF (as indicated in thefigure captions). For analyses carried out using transiently expressingcells, plasmid DNA encoding a constitutively expressed fluorescentprotein marker was co-delivered at the time of transfection and used toidentify positively transfected cells populations (see SupplementaryFIG. 12). Briefly, transfected cells were were gated to the top 1% ofmarker fluorescence of non-transfected control cells under the samecondition. Transient expression experiments carried out using the“turn-on” TFs (shown in FIGS. 1d and 1i , and Supplementary FIG. 3) weregated via detection of an mCherry marker that was expressed via an IRESsequence on the TF-encoding plasmid. Transfected cells were incubatedfor 24-48 h after transfection either in the presence or absence of theindicated NS3 inhibitors before being analysed using an Attune NxT flowcytometer (ThermoFisher). For the analyses in which rTetR-NS3-VP64-p65and TMD-NS3-Gal4min were combined, 125,000 HEK 293FT cells weretransfected with 25 ng of DNA (per-125,000 cells) with DNA mixturescontaining a 3:3:2:2 molar ratio of rTetR-NS3-VP64-p65 to TMD-NS3-Gal4to TRE mTagBFP to UAS H2B Citrine (as approximated by DNA size).

For expression analyses carried out using dCas9-based TFs, HEK 293FTcells were seeded in 48-well plates and transfected at a density of−80,000 cells per well. A total 250 ng of DNA was delivered per well ineach experiment; sgRNA- and dCas9-encoding constructs were transfectedat a 1:1 molar ratio as approximated based on DNA size. The constructsencoding the sgRNAs targeting human CXCR4 promoter were acquired fromAddGene and were previously reported (“sgC2” and “sgC3”). Expression ofthe chemokine receptor was analysed via flow cytometry using afluorescently-conjugated CXCR-4 antibody; geometric means were recorded.

Statistics and Reproducibility

All flow cytometry and luminescence assay data were collected using 3biologically independent samples. For fluorescence imaging analyses, ≥3images per condition were recorded (encompassing hundreds of cells), andrepresentative images are displayed in the figures. For immunoblotting,analyses were repeated ≥3 times and a representative blots were chosenfor display.

Bulky Ectodomain Mechanoreceptor Embodiment

Gamma secretase is a membrane protein complex involved in biologicalfunctions such as Notch and amyloid precursor protein (APP) processing.Its proteolytic subunit, presenilin, acts by catalyzing the cleavage ofintramembrane alpha helices, and in turn allows the release of both theextracellular domain (important in APP pathology) and the intracellulardomain (important in Notch developmental biology). The gamma secretaseextracellular subunit, nicastrin, has been shown to regulate thisprocess through steric hindrance. Gamma secretase substrates with bulkyextracellular domains are resistant to proteolysis, and the regulatedshedding of this bulky ectodomain is a key pathway in Notch processing.

Shown here in FIG. 30 are the results using a novel mechanoreceptor witha fluorescent protein as its bulky ectodomain. Green fluorescent proteinhas been shown to unfold at approximately 100 pN (Dietz 2004), andunfolding of this domain would reduce steric hindrance and in turn allowthe release of an intracellular transcription factor through gammasecretase processing.

Cells transiently transfected with DNA encoding this receptor displayincreased activation when plated on wells coated with an antibody thatbinds it. However, this increase in activation only occurred when theantibodies are tethered, and thus are able to apply force to thereceptors: soluble antibodies at similar concentrations did not increaseactivation. Additionally, this process is supported to be gammasecretase dependent, as addition of a gamma secretase inhibitordiminished cell activation.

Wells from an untreated 96 well plate were coated overnight (20 hrs)with 0.005% gelatin and with or without 1 μg of an anti-myc antibody.They were then washed 3 times with 2004 PBS, and reporter cells that hadbeen transfected with a plasmid encoding this protein were plated withmedia (10% FBS No antibiotics) with or without DAPT, a gamma secretaseinhibitor (GS inhibitor). 2 hours later, anti-myc antibody was added towells, and the cells were cultured overnight (20 hrs) at 37 degreesCelsius. All results are from flow cytometry, gated for live GFP+ cells.3 samples for each condition.

REFERENCES

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Example 3

Reduction of SynNotch Leakiness by Incorporation of the JuxtamembraneLWF Motif.

SynNotch receptors are designed around a preserved Notch regulatory“core,” the minimal protein unit identified as necessary for maintainingthe natural Notch signaling mechanism. Specifically, SynNotch coresretain the Negative Regulatory Region (NRR) and Transmembrane Domain(TMD) of natural Notch. Although SynNotch receptors successfully mimicmany key Notch signaling characteristics, they fail to recapitulate someregulatory aspects of Notch signaling; natural Notch signaling istightly regulated, but SynNotch signaling is often observed as noisy or“leaky,” with some SynNotch constructs having significant backgroundactivation even in the absence of stimulus.

The leakiness of SynNotch receptors may be due the introduction ofsynthetic protein modules, the absence of natural Notch components, orlikely some combination thereof. For example, it has been observed thataddition of more powerful transcriptional activators (VPR, as opposed toVP64) leads to an extreme increase in background noise and renders thereceptors near inviable.

Pursuing the hypothesis that the SynNotch regulatory core may be missingan element of the natural receptor important for regulating signalingdynamics, Notch's “LWF motif” was identified as a candidate forreintroduction into SynNotch. The LWF motif, absent in SynNotch, is ahydrophobic stretch of amino acids juxtamembrane to the TMD's C-terminusthat was recently modeled to briefly reenter the cytosoloic face of thecell membrane [1] (FIG. 31A). The TMD of Notch contains the S3 site forcleavage by γ-secretase, the final proteolytic event necessary forreceptor activation. Dysregulated cleavage by γ-secretase is a likelycause of SynNotch leakiness, as indicated by the ability of DAPT(γ-secretase inhibitor) to suppress leaky receptor activation. Due tothe LWF motif's proximity with the TMD and S3, as well as modeledinteractions with the cell membrane, without wishing to be bound by aparticular theory, it was hypothesized that inclusion of the motif couldrestore some natural regulation of receptor activity. Because themodeled LWF motif structure was not published until after SynNotch, itwas likely not considered in the original SynNotch core design.

Data herein provides preliminary evidence that inclusion of the LWFmotif reduces background activation in SynNotch receptors. In initialexperiments, the original SynNotch core design (SN) was compare with aSynNotch core containing the LWF motif (SN-LWF) (FIG. 31A). Eachreceptor core was test with two different transcription factorintracellular domains (ICD's): Gal4 fused to VP64 (“VP64,” a modesttranscriptional activator commonly used in SynNotch) and Gal4 fused toVPR (“VPR,” a strong transcriptional activator, whose strength ispenalized by high background activation in SynNotch constructs). ForVP64 ICD's, it was found that both SN-VP64 and SN-LWF-VP64 have lowbackground activation and activate to the same extent when stimulated,as expected for the modest transcriptional activator (FIG. 31B). For thestronger VPR ICD, however, it was found that SN-VPR is significantlyleakier than SN-LWF-VPR, while both activate to comparable extents (FIG.31C). These data indicate that inclusion of the LWF motif in SynNotchmay permit the use of valuable transcriptional effectors that are notviable in leaky settings. For example, if tightly regulated like naturalNotch signaling, potent domains such as VPR could allow more rapidtranscriptional outputs in therapeutic settings, or DNA editors such asCre and Cas9 could be used without risk of erroneous gene editing.

REFERENCE

-   [1] Deatherage et al. “Structural and biochemical differences    between the Notch and the amyloid precursor protein transmembrane    domains.” Science Advances, 2017.

Example 4

Previously, a design strategy and preliminary data for SynNotchreceptors with programmable force-activation thresholds was introduced.Natural and synthetic Notch receptors contain a Negative RegulatoryRegion (NRR), which is an auto-inhibitory domain that opens in responseto tensile forces greater than ˜5 pN. This mechanical opening of the NRRis necessary for downstream transcriptional activity of the receptor. Inorder to create Notch-based receptors with force-activation thresholdsabove 5 pN, an scFv targeting the NRR was fused to the N-terminus of theNRR itself. Receptors containing this scFv-NRR (“sNRR”) domain requiregreater tensile force for activation due to the additional proteininteraction that must be disrupted to open the NRR. Furthermore, theprecise amount of force required for receptor activation is determinedby scFv binding affinity for the NRR and is thus tunable.

Provided herein are additional embodiments of the syntheticmechanoreceptors, their sNRR domains, and the potential applications ofthe invention described herein. In certain embodiments, the designcriteria for building functional mechanoreceptors include, but are notlimited to: they must be expressed at the cell surface (FIGS. 32A and32B), they must activate in response to physiologically relevant andmeasurable forces (FIGS. 33A and 33B), and this force-activationthreshold must be tunable (FIG. 34A-34E).

FIG. 35 shows that SynNotch receptors containing a sNRR domain expressedat the surface of HeLa cells similarly to receptors containing the WTNRR domain. Data presented in FIG. 35 indicate that the receptors can bestably expressed in HEK 293FT cells.

Previously herein, the use of a tension gauge tether (TGT) assay toquantify mechanostability in the engineered receptors described hereinwas introduced. FIGS. 33A and 33B show evidence for the increasedmechanical strength of the sNRR domain described herein. While the WTNRR opens in response to ˜5 pN of tensile force, the sNRR domain has atension tolerance closer to ˜50 pN.

Next, it was set out to tune the mechanical strength of sNRR and createa collection of receptors that activate in response definable andphysiologically relevant forces. Because the unbinding force of anantibody-antigen pair correlates with its thermal dissociation rate, itwas hypothesized that sNRR tension tolerance could be altered bymutating affinity of the scFv for the NRR. Previously presented datashows proof-of-concept evidence for this approach, using two model pointmutations on the scFv, which was expressed as a separate transmembranecis-inhibitor. In FIG. 34A-34E presents further evidence supporting thisdesign strategy. A collection of mutated sNRR domains exhibiting aspectrum of mechanical strengths was generated. Notably, the impact ofresidue mutation on receptor strength followed predictable trends; moresevere point mutations resulted in weaker receptors (Y49A weaker thanY49F, R99A weaker than R99K), and double point mutations were additivelyweaker than their constituents. Time-course microscopy of cellsexpressing mutated sNRR receptors further demonstrates the receptors'distinct mechanical characteristics, as well as their ability todiscriminate between mechanical stimuli over time. Lastly, providedherein is a mathematical model that captures the observed temporaltrends in receptor activation, simply by varying (1) the scFv-NRRdissociation rate vs. (2) the dissociation rate of the TGT doublestranded DNA. The ability of such a simple two-parameter model torecapitulate our observed results indicates that mutating scFv-NRRaffinity is indeed sufficient to create mechanically distinct receptors.

Importantly, the mechanoreceptors presented herein not only measure anapplied force, but respond to and make a decision based off that force.In various embodiments, designer responses to force may be therapeuticin nature, or geared to study/recapitulate natural phenomenon inmechanobiology. For example, stem cells have been shown to differentiatedown lineages dictated by their mechanical environment. Presented hereinin FIG. 35, this natural process was mimic by using sNRR-based SynNotchto drive myogenic differentiation of fibroblast cells based onmechanical TGT stimulus.

Lastly, data presented herein show that the choice of NRR-binding scFvis nontrivial in the design of sNRR (FIG. 36). Three additionalantibodies known to bind the NRR and inhibit Notch activation wereincorporated into sNRR domains as scFv's. Although known to be effectiveas soluble antibodies, these scFv's offer little to no additionalmechanical stability to the receptor. The original sNRR was designed toaccommodate binding of the particular scFv in use (based off the crystalstructure of the antibody-antigen complex), and incorporation ofdifferent scFv's would require additional engineering to optimallyposition them with respect to the NRR.

Sequences

Provided herein are sequences for use, in whole or in part, in thevarious embodiments of the compositions, methods, and systems describedherein.

SEQ ID NO: 1 is an amino acid sequence encoding signal Sequence (SS).

(SEQ ID NO: 1) AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRR T

SEQ ID NO: 2 is an amino acid sequence encoding human Notch1 LBD.

(SEQ ID NO: 2) RGPRCSQPGETCLNGGKCEAANGTEACVCGGAFVGPRCQDPNPCLSTPCKNAGTCHVVDRRGVADYACSCALGFSGPLCLTPLDNACLTNPCRNGGTCDLLTLTEYKCRCPPGWSGKSCQQADPCASNPCANGGQCLPFEASYICHCPPSFHGPTCRQDVNECGQKPGLCRHGGTCHNEVGSYRCVCRATHTGPNCERPYVPCSPSPCQNGGTCRPTGDVTHECACLPGFTGQNCEENIDDCPGNNCKNGGACVDGVNTYNCRCPPEWTGQYCTEDVDECQLMPNACQNGGTCHNTHGGYNCVCVNGWTGEDCSENIDDCASAACFHGATCHDRVASFYCECPHGRTGLLCHLNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPACSQDVDECSLGANPCEHAGKCINTLGSFECQCLQGYTGPRCEIDVNECVSNPCQNDATCLDQIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRCLDKINEFQCECPTGFTGHLCQYDVDECASTPCKNGAKCLDGPNTYTCVCTEGYTGTHCEVDIDECDPDPCHYGSCKDGVATFTCLCRPGYTGHHCETNINECSSQPCRHGGTCQDRDNAYLCFCLKGTTGPNCEINLDDCASSPCDSGTCLDKIDGYECACEPGYTGSMCNINIDECAGNPCHNGGTCEDGINGFTCRCPEGYHDPTCLSEVNECNSNPCVHGACRDSLNGYKCDCDPGWSGTNCDINNNECESNPCVNGGTCKDMTSGYVCTCREGFSGPNCQTNINECASNPCLNQGTCIDDVAGYKCNCLLPYTGATCEVVLAPCAPSPCRNGGECRQSEDYESFSCVCPTGWQAGQTCEVDINECVLSPCRHGASCQNTHGGYRCHCQAGYSGRNCETDIDDCRPNPCHNGGSCTDGINTAFCDCLPGFRGTFCEEDINECASDPCRNGANCTDCVDSYTCTCPAGFSGIHCENNTPDCTESSCFNGGTCVDGINSFTCLCPPGFTGSYCQHDVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQNLVHWCDSSPCKNGGKCWQTHTQYRCECPSGWTGLYCDVPSVSCEVAAQRQGVDVARLCQHGGLCVDAGNTHHCRCQAGYTGSYCEDLVDECSPSPCQNGATCTDYLGGYSCKCVAGYHGVNCSEEIDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHCEINVDDCNPPVDPVSRSPKCFNNGTCVDQVGGYSCTCPPGFVGERCEGDVNECLSNPCDARGTQNCVQRVNDFHCECRAGHTGRRCESVINGCKGKPCKNGGTCAVASNTARGFICKCPAGFEGATCENDARTCGSLRCLNGGTCISGPRSPTCLCLGPFTGPECQFPASSPCLGGNPCYNQGTCEPTSESPFYRCLCPA KFNGLLCH

SEQ ID NO: 3 is an amino acid sequence encoding LaG17 anti-GFP nanobody.

(SEQ ID NO: 3) MADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVS

SEQ ID NO: 4 is an amino acid sequence encoding scFv αFITC.

(SEQ ID NO: 4) QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYAGHFYSYMDVWGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL G

SEQ ID NO: 5 is an amino acid sequence encoding SNAP tag.

(SEQ ID NO: 5) MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLG

SEQ ID NO: 6 is an amino acid sequence encoding FLAG tag.

(SEQ ID NO: 6) DYKDDDDKG

SEQ ID NO: 7 is an amino acid sequence encoding Myc tag.

(SEQ ID NO: 7) EQKLISEEDL

SEQ ID NO: 8 is an amino acid sequence encoding human NRR1.

(SEQ ID NO: 8) ILDYSFGGGAGRDIPPPLIEEACELPECQEDAGNKVCSLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQRAEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFPYYGREEELRKHPIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRELDPMDVRGSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSETV EPPPPAQ

SEQ ID NO: 9 is an amino acid sequence encoding mouse NRR1.

(SEQ ID NO: 9) ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQ

SEQ ID NO: 10 is an amino acid sequence encoding mouse NRR 2.

(SEQ ID NO: 10) LYTAPPSTPPATCLSQYCADKARDGVCDEACNSHACQWDGGDCSLTMENPWANCSSPLPCWDYINNQCDELCNTVECLFDNFECQGNSKTCKYDKYCADHFKDNHCDQGCNSEECGWDGLDCAADQPENLAEGTLVIVVLMPPEQLLQDARSFLRALGTLLHTNLRIKRDSQGELMVYPYYGEKSAAMKKQRMTRRSLPGEQEQEVAGSKVFLEIDNRQCVQDSDHCFKNTDAAAALLASHAIQGTLSYP LVSVVSESLTPERTQ

SEQ ID NO: 11 is an amino acid sequence encoding NICD.

(SEQ ID NO: 11) QHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLETKKFRFEEPVVLPDLDDQTDHRQWTQQHLDAADLRMSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQDNMGRTPLHAAVSADAQGVFQILIRNRATDLDARMHDGTTPLILAARLAVEGMLEDLINSHADVNAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNREETPLFLAAREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGAPLGGTPTLSPPLCSPNGYLGSLKPGVQGKKVRKPSSKGLACGSKEAKDLKARRKKSQDGKGCLLDSSGMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSVPLNHLPGMPDTHLGIGHLNVAAKPEMAALGGGGRLAFETGPPRLSHLPVASGTSTVLGSSSGGALNFTVGGSTSLNGQCEWLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAASALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPPPPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSLVPPVTAAQFLTPPSQHSYSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSDWSEGVSSPPTSMQSQIARIPEAFK

SEQ ID NO: 12 is an amino acid sequence encoding Gal4DBD-VP64.

(SEQ ID NO: 12) MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDA LDDFDLDMLGS

SEQ ID NO: 13 is an amino acid sequence encoding human Notch1 TM.

(SEQ ID NO: 13) LHFMYVAAAAFVLLFFVGCGVLLSRKRRR

SEQ ID NO: 14 is an amino acid sequence encoding mouse Notch1 TM.

(SEQ ID NO: 14) LHLMYVAAAAFVLLFFVGCGVLLSRKRRR

SEQ ID NO: 15 is an amino acid sequence encoding Ab2; wherein in SEQ IDNO: 15, bold text depicts the VH domain, underlined text depicts thelinker domain, and double underlined text depicts the VL domain).

(SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGS GFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGG DIOMTOSPSSLSASVGDRVTITCRASODVSTAVAWYOOKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

The amino acid sequences of SEQ ID NO:16-24, depict amino acid changesfrom SEQ ID NO:15

SEQ ID NO: 16 is an amino acid sequence encoding Ab2-VH N55A. The VH N55mutation is depicted in bold, underlined text.

(SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVAR INPPA RSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 17 is an amino acid sequence encoding Ab2-VH R99K. The VH R55mutation depicted in bold, underlined text.

(SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGS GF KWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 18 is an amino acid sequence encoding Ab2-VH R99A. The VH R99mutation is depicted in bold, underlined text.

(SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGS GF AWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 19 is an amino acid sequence encoding Ab2-VH C22S/C92A. TheVH C22 and C92 mutations are depicted in bold, underlined text.

(SEQ ID NO: 19) EVQLVESGGGLVQPGGSLRLSSAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY A ARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 20 is an amino acid sequence encoding Ab2-VL 530A. The VL S30mutation is depicted in bold, underlined text.

(SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSA SVGDRVTITCRASQDV ATAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 21 is an amino acid sequence encoding Ab2-VL Y49A. The VL Y49mutation is depicted in bold, underlined text.

(SEQ ID NO: 21) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLI A SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 22 is an amino acid sequence encoding Ab2-VL Y55A. The VL Y55mutation is depicted in bold, underlined text)

(SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL A SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 23 is an amino acid sequence encoding Ab2-VL F91A. The VL F91mutation is depicted in bold, underlined text.

(SEQ ID NO: 23) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ A YTTPSTFGQGTKVEIK

SEQ ID NO: 24 is an amino acid sequence encoding Ab2-VL Y92A. The VL Y92mutation shown in bold, underlined text.

(SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQF A TTPSTFGQGTKVEIK

SEQ ID NO: 25 is an amino acid sequence encoding E6.

(SEQ ID NO: 25) QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYAGHFYSYMDVWGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL G

SEQ ID NO: 26 is an amino acid sequence encoding WC629.

(SEQ ID NO: 26) EVQLVQSGAEVKKPGSSVKVSCKASGGTLSSYTVSWLRQAPGQGLEWMGRIIPILDRANYAQKFQGRVTITADKSTSTAYMELNSLRSDDTAVYYCARSIGAAGDGVWFDPWGQGTMVTVSSGGSSRSSSSGGGGSGGGGQAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIFDNKNRPSGVPDRFSGSNSGTSASLAITGLQAEDEAEYYCQSYDNNLSGRVFGGGTKLTV

SEQ ID NO: 27 is an amino acid sequence encoding WC75.

(SEQ ID NO: 27) LVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSSISWHSRTIAYADSVKGRFSISRDNAKNSLYLQMNSLRPEDTAVYYCAKASYLSTSSSLDYWGRGTLVTVSSGGSSRSSSSGGGGSGGGGQSVLTQPGSVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL

SEQ ID NO: 28 is an amino acid sequence encoding D3.

(SEQ ID NO: 28) EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVRQAPGKGLEWVSYIYPYSGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHSGYYRISSAMDVWGQGTLVTVSAGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQNIKRFLAWYQQKPGKAPKLLIYGASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRSPHTFGQGTKVE IKRGG

SEQ ID NO: 29 is an amino acid sequence encoding B6.

(SEQ ID NO: 29) AQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWMNAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDRVPTIPAYRIDYWGQGTLVTVSSLEGGGGSGGGGSGGGASDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPRLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKLEIKR

SEQ ID NO: 30 is an amino acid sequence encoding B9.

(SEQ ID NO: 30) AQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGPRSYGAGGMDVWGQGTLVTVSSLEGGGGSGGGGSGGGASDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKR

SEQ ID NO: 31 is an amino acid sequence encoding Platelet Derived GrowthFactor Receptor (PDGFR) TM domain.

(SEQ ID NO: 31) AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRR T

SEQ ID NO: 32 is an amino acid sequence encoding NS3. In SEQ IC NO:32,the bold, underlined text depicts the N- and C-terminal cis-cleavagesites and AU1 tag.

(SEQ ID NO: 32) EDVVCCHSIYGKKKGDI DTYRYI GSSGTGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDRE VLYQEFDEMEECSQH

SEQ ID NO: 33 is an amino acid sequence encoding AU1 tag.

(SEQ ID NO: 33) DTYRYI

SEQ ID NO: 34 is an amino acid sequence encoding rat Dll1 SS-ECD.

(SEQ ID NO: 34) MGRRSALALAVVSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGSGPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTAVLGVDSFSLPDGAGIDPAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLTTQRHLTVGEEWSQDLHSSGRTDLRYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKMCDPGWKGQYCTDPICLPGCDDQHGYCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCRNGATCTNTGQGSYTCSCRPGYTGANCELEVDECAPSPCRNGGSCTDLEDSYSCTCPPGFYGKVCELSAMTCADGPCFNGGRCSDNPDGGYTCHCPAGFSGFNCEKKIDLCSSSPCSNGAKCVDLGNSYLCRCQTGFSGRYCEDNVDDCASSPCANGGTCRDSVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHQRGQRYMCECAQGYGGANCQFLLPEPPPDLIVAAQGGSFPW

SEQ ID NO: 35 is an amino acid sequence encoding rat Dll1 TM domain andICD.

(SEQ ID NO: 35) VAVCAGVVLVLLLLLGCAAVVVCVRLKLQKHQPPPDPCGGETETMNNLANCQREKDVSVSIIGATQIKNTNKKADFHGDHGADKSSFKARYPTVDYNLIRDLKGDEATVRDAHSKRDTKCQSQGSVGEEKSTSTLRGGEVPDRKRPESVYSTSKDTKYQSVYVLSAEKDECVIATEV

SEQ ID NO: 36 is an amino acid sequence encoding human Dll4 SS-ECD.

(SEQ ID NO: 36) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPW

SEQ ID NO: 37 is an amino acid sequence encoding human Dll4 TM domainand ICD.

(SEQ ID NO: 37) VAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNEC VIATEV

SEQ ID NO: 38 is an amino acid sequence encoding mouse Dll4 SS-ECD.

(SEQ ID NO: 38) MTPASRSACRWALLLLAVLWPQQRAAGSGIFQLRLQEFVNQRGMLANGQSCEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSGRNPLQLPFNFTWPGTFSLNIQAWHTPGDDLRPETSPGNSLISQIIIQGSLAVGKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDHFGHYECQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECICRPGWQGRLCNECIPHNGCRHGTCSIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGSTCSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCLCPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNCEKKVDRCTSNPCANGGQCQNRGPSRTCRCRPGFTGTHCELHISDCARSPCAHGGTCHDLENGPVCTCPAGFSGRRCEVRITHDACASGPCFNGATCYTGLSPNNFVCNCPYGFVGSRCEFPVGLPPSFPWVA

SEQ ID NO: 39 is an amino acid sequence encoding mouse Dll4 TM domainand ICD.

(SEQ ID NO: 39) VSLGVGLVVLLVLLVMVVVAVRQLRLRRPDDESREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKLQNHTLDYNLAPGLLGRGGMPGKYPHSDKSLGEKVPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVI ATEV

SEQ ID NO: 40 is an amino acid sequence encoding mCherry.

(SEQ ID NO: 40) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

SEQ ID NO: 41 is an amino acid sequence encoding SS for cis-inhibitors.METDTLLLWVLLLWVPGSTGDGGGG (SEQ ID NO: 41)

SEQ ID NO: 42 is an amino acid sequence encoding an exemplary scFv(depicted in bold text) is expressed within a SynNotch as an “LNR4”domain. The underlined text depicts the linker, the double underlined,italic text depicts the linker with myc tag.

(SEQ ID NO: 42) GGGGSTGDGGGG EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQG TKVEIKGGGSEQKLISEEDLGGGS

SEQ ID NO: 43 is an amino acid sequence encoding an exemplary fullconstruct for a mouse-based SynNotch with myc-tagged LBD that can bindFITC or GFP, an Ab2 LNR4, and a Gal4-VP64 ICD. SEQ ID NO: 43 comprises,from N to C, a Signal Sequence; LBD (which can comprise multiple domainsto bind different ligands; depicted in bold text); Notch Core (whichcomprises a Notch1 NRR, and an optional LNR4 domain expressed N-terminalto the NRR, depicted in underlined text); TM domain (depicted initalicized text); and an ICD (depicted in bold, underlined text).

(SEQ ID NO: 43) MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYAGHFYSYMDVVVGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGSMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTG TAFDYWGQGTQVTVSGGGGSTGDGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIKGGGSEQKLISEEDLGGGSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLL FFVGCGVLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS

SEQ ID NO: 44 is an amino acid sequence encoding an exemplary scFvexpressed as a separate transmembrane cis-inhibitor of Notch. SEQ ID NO:44 comprises, from N to C, Signal Sequence; scFV (depicted in boldtext); Myc Tag and TM domain (depicted in italicized text); and mCherry(depicted in underlined text).

(SEQ ID NO: 44) METDTLLLWVLLLWVPGSTGDGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFYTTPSTFGQGTKVEIKGGGSEQKLISEEDLGGGSAVGQDTQEVIVVP HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRRTMVSKGEEDNMAI IKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

SEQ ID NO: 45 is an amino acid sequence encoding an exemplary fullDLL1-NS3 construct (rat Dll1) with C-terminal mCherry. SEQ ID NO: 45comprises, from N to C, Signal sequence, Dll1 ECD; T7 tag (depicted inbold text); NS3; HA Tag (depicted in italicized text); TM domain; Dll1ICD; and mCherry (depicted in underlined text.)

(SEQ ID NO: 45) MGRRSALALAVVSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGSGPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTAVLGVDSFSLPDGAGIDPAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLTTQRHLTVGEEWSQDLHSSGRTDLRYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKMCDPGWKGQYCTDPICLPGCDDQHGYCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCRNGATCTNTGQGSYTCSCRPGYTGANCELEVDECAPSPCRNGGSCTDLEDSYSCTCPPGFYGKVCELSAMTCADGPCFNGGRCSDNPDGGYTCHCPAGFSGFNCEKKIDLCSSSPCSNGAKCVDLGNSYLCRCQTGFSGRYCEDNVDDCASSPCANGGTCRDSVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHQRGQRYMCECAQGYGGANCQFLLPEPPPDLIVAAQGGSFPWSRADMASMTGGQQMGSTEDVVCCHSIYGKKKGDIDTYRYIGSSGTGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDREVLYQEFDEMEECSQHYPYDVPDYAGASAVAVCAGVVLVLLLLLGCAAVVVCVRLKLQKHQPPPDPCGGETETMNNLANCQREKDVSVSIIGATQIKNTNKKADFHGDHGADKSSFKARYPTVDYNLIRDLKGDEATVRDAHSKRDTKCQSQGSVGEEKSTSTLRGGEVPDRKRPESVYSTSKDTKYQSVYVLSAEKDECVIATEVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKS

SEQ ID NO: 50 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(Gal4).DB_(Gal4) is in bolded text, TA_(Gal4) is in underlined text, NS4A is initalicized text, NS3 is in bolded, underlined text, and NS3 Cut Site isin bolded, italicized text.

(SEQ ID NO: 50) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTgggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACG

CGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGG ATCCGGCACTAGTGCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTT

T ATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCATGCGCCAACTTTAATCAAAGTGGAAACATCGCGGACAGCTCACTCAGCTTTACCTTCACCAATAGCAGTAACGGGCCGAACCTCATAACCACCCAGACCAACAGCCAGGCCTTGAGCCAGCCGATCGCCTCATCTAACGTGCATGATAACTTTATGAACAACGAGATCACCGCGAGTAAGATAGACGACGGGAACAACAGCAAGCCCCTTAGCCCAGGTTGGACGGACCAGACCGCCTACAACGCTTTCGGCATTACGACCGGCATGTTCAACACCACGACCATGGACGATGTGTACAACTACCTGTTCGATGACGAAGACACACCGCCAAACCCCAAAAAAGAA

SEQ ID NO: 51 is a nucleotide sequence encoding DB_(Gal4)-NS3-TAVP32.DBGal4 is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3Cut Site is in bold/italics, NLS underlined, VP32 is inbold/italics/underlined

(SEQ ID NO: 51) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTgggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACG

CGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGG ATCCGGCACTAGTGCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTT

T ATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGAC GGGCT

SEQ ID NO: 52 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(VP64).DB_(Gal4) is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3Cut Site is in bold/italics, NLS is underlined, and VP64 is inbold/italics/underlined.

(SEQ ID NO: 52) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTgggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACG

CGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGG ATCCGGCACTAGTGCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTT

T ATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGAC GGGCT

SEQ ID NO: 53 is a nucleotide sequence encodingDB_(Gal4)-NS3-TA_(VP64-p65). DB_(Gal4) is in bold, NS4A is in italics,NS3 is in bold/underlined, NS3 Cut Site is in bold/italics, NLS isunderlined, VP64 is in bold/italics/underlined, P65 is doubleunderlined.

(SEQ ID NO: 53) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTgggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACGGAGGACGTGGTGTGCTGCCACTCAATCTACGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCC AGCGGTTCCGGACGGGCT

ATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCT TGCTG

SEQ ID NO: 54 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(VPR)DB_(Gal4) is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3Cut Site is in bold/italics, NLS is underlined, VP64 is inbold/italics/underlined, P65 in double underlined, Rta in bold, doubleunderlined.

(SEQ ID NO: 54) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTgggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACG

CGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCC GGATCCGGCACTAGTGCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTT

CTATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTT CCGGACGGGCT

ATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTGGG CTCTGGCAGCGGCAGCCGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCATTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAGAAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACCACACTTGAGTCCATGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGACTGTCCATCTTCGACACATCTCTGTTT

SEQ ID NO: 55 is a nucleotide sequence encodingDB_(rTetR)-NS3-TA_(VP64-p65). rTet in bold, NS4A in italics, NS3 inbold/underlined, NS3 Cut Site in bold/italics, NLS underlined, VP64 inbold/italics/underlined, and P65 is double underlined.

(SEQ ID NO: 55) ATGTCTAGACTGGACAAGAGCAAAGTCATAAACGGAGCTCTGGAATTACTCAATGGTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCgggGCGTCTGCAggcATGGCCAGCATGACTGGTGG ACAGCAAATGGGGTCGACG

GGCAAGAAGAAGGGTGA TATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACT CCTCTCCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGG GAGGTT

TATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCT

ATTA ACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTG

SEQ ID NO: 56 is a nucleotide sequence encoding TMD-NS3-Gal4_(min).Signal Sequence in double underlined/italics,

,

, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics,DB_(Gal4) in bold, and

(SEQ ID NO: 56)ATGGAGACCGACACCCTGCTCCTGTGGGTGTTGTTGCTCTGGGTCCCAGGTTCTACCGGC

TGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTTCTCTACCAGGAGTTCGATGAGATGGAAGAGTGCTCTCAGCACTATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCATGCGGTACCATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGC

SEQ ID NO: 57 is a nucleotide sequence encodingBFP-TMD-NS3-Gal4_(min)-mCherry. Signal Sequence in doubleunderlined/italics, BFP in bold/dashed underlined,

, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics,DB_(Gal4) in bold,

, and

.

SEQ ID NO: 57)

ATCGACACCTATAGATATATCGGAAGTAGTGGGACCGGCTGCGTGGTGATCGTAGGGCGGATAGTGCTGTCTGGCTCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCATCTGCATGCGGTACCATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAA

SEQ ID NO: 58 is a nucleotide sequence encoding myr-palm-NS3-Gal4_(min).Myr-palm in bold/italics/underlined, NS4A in italics, NS3 inbold/underlined, NS3 Cut Site in bold/italics, DB_(Gal4) in bold,TA_(Gal4) is double underlined.

(SEQ ID NO: 58)

CGTACGATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACG

GGCAAGAAGAAGG GTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGAC AACTCCTCTCCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGA TAGGGAGGTT

TATCCCTACGATGTGCCCG ATTACGCTGGCGCGTCTGCATGCGGTACCATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGCTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTGCCAACTTTAATCAAAGTGGAAACATCGCGGACAGCTCACTCAGCTTTACCTTCACCAATAGCAGTAACGGGCCGAACCTCATAACCACCCAGACCAACAGCCAGGCCTTGAGCCAGCCGATCGCCTCATCTAACGTGCATGATAACTTTATGAACAACGAGATCACCGCGAGTAAGATAGACGACGGGAACAACAGCAAGCCCCTTAGCCCAGGTTGGACGGACCAGACCGCCTACAACGCTTTCGGCATTACGACCGGCATGTTCAACACCACGACCATGGACGATGTGTACAACTACCTGTTCGATGACGAAGACACACCGCCAAAC CCCAAAAAAGAA

SEQ ID NO: 59 is a nucleotide sequence encodingTMD-NS3-DB_(Gal4)-TA_(VP64). Signal Sequence in italics/doubleunderlined, scfv linker in bold/double underlined, TMD in zigzagunderlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site inbold/italics, DB_(Gal4) in bold, VP64 is dotted underlined.

(SEQ ID NO: 59)ATGGAGACCGACACCCTGCTCCTGTGGGTGTTGTTGCTCTGGGTCCCAGGTTCTACCGGCGACGGAGGAGGCGGCGAAGTACAGCTGGTGGAGTCCGGCGGTGGCCTCGTGCAACCCGGAGGGTCCTTGAGGCTGTCCTGTGCAGCCAGCGGTTTCACGTTCAGCAGCTACTGGATTCACTGGGTGAGGCAAGCTCCGGGCAAGGGCCTGGAGTGGGTTGCGAGGATAAACCCCCCCAACAGGTCCAACCAGTACGCCGATAGCGTGAAGGGTCGGTTCACCATCAGCGCCGACACTAGCAAGAACACGGCCTACCTGCAGATGAACTCTCTGAGGGCCGAGGACACAGCGGTGTACTATTGCGCCAGGGGCTCAGGGTTTCGATGGGTCATGGATTACTGGGGCCAAGGCACCCTGGTTACCGTTTCTAGCGGCGGATCTAGCAGGAGCTCATCATCCGGGGGTGGCGGGAGCGGAGGTGGTGGGGATATTCAGATGACGCAATCTCCGTCCTCCCTCAGCGCAAGCGTGGGCGACAGGGTGACCATTACTTGTCGCGCCTCTCAGGATGTGAGCACTGCTGTGGCCTGGTATCAACAAAAACCCGGCAAAGCCCCCAAACTGCTGATCTACTCTGCCAGCTTTCTGTACTCAGGCGTGCCCAGCAGGTTTTCCGGCTCCGGCAGCGGCACCGACTTCACCCTTACCATTAGCAGCCTG

TACGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGG

GTGCCCGATTACGCTGGCGCGTCTGCATGCGGTACCATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAAGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAGAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACAACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGCAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGAGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGC

SEQ ID NO: 60 is a nucleotide sequence encoding dCas9-NS3-NLS-VPR. dCas9in bold/italics/underlined, NS4A in italics, NS3 in bold/underlined, NS3Cut Site in bold/italics, NLS in bold, VP64 is double underlined, P65 indouble underlined/italics,

(SEQ ID NO: 60)

GATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGTG CGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTTCTCTACCAGGAGTTCGATGAGATGGAAGAGTGCTCTCAGCACTATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCC

SEQ ID NO: 61 is a nucleotide sequence encoding dCas9-NS3-NLS-VPR.

, dCas9 in bold/italics/underlined, NS4A in italics, NS3 inbold/underlined, NS3 Cut Site in bold/italics, NLS in bold, VP64 isdouble underlined, P65 in double underlined/italics, Rta is bold/dottedunderlined.

(SEQ ID NO: 61)

GCGTGGTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGTGCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCTCCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTTCTCTACCAGGAGTTCGATGAGATGGAAGAGTGCTCTCAGCACTATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGG

SEQ ID NOs 62-64 are nucleotide sequences encoding sgRNAs, e.g., usedherein. These sequences are further described in Zalatan et al. Cell.2015., Nihongaki et al., Nature Chem Bio. 2015, which are incorporatedherein by reference in their entireties.

sgC2¹ SEQ ID NO: 62 GCAGACGCGAGGAAGGAGGGCGC sgC3¹ SEQ ID NO: 63GCCTCTGGGAGGTCCTGTCCGGCTC GAL4 sgRNA1² SEQ ID NO: 64TGGGTCTTCGGAGGACAGTACTC

SEQ ID NO: 65 is a nucleotide sequence encoding NS3-Dll1-mCherry. Signalsequence in bold, Rat Dll1 ECD in italics, NS3 cut site underlined, NS4Ain bold/italics, NS3 in bold/underlined, Rat Dll1 TMD/ICD inbold/italics/underlined, mCherry in double underlined.

(SEQ ID NO: 65) ATGGGCCGTCGGAGCGCTCTAGCCCTTGCCGTGGTCTCAGCCCTGCTGTG CCAGGTCTGGAGCTCTGGCGTATTTGAGCTGAAGCTGCAGGAGTTCGTCAACAAGAAGGGGCTGCTGGGGAACCGCAACTGCTGCCGCGGGGGCTCTGGCCCGCCGTGCGCCTGCAGGACCTTCTTTCGCGTATGCCTCAAGCATTACCAGGCCAGCGTGTCCCCGGAGCCACCCTGCACCTACGGCAGTGCGGTCACCGCAGTGCTGGGTGTCGACTCCTTCAGCCTGCCTGATGGCGCAGGCATCGACCCCGCCTTCAGCAACCCCATCCGATTCCCCTTCGGATTCACCTGGCCAGGTACCTTCTCTCTGATCATTGAAGCCCTCCACACAGATTCTCCTGACGACCTCGCAACAGAAAACCCAGAAAGACTCATCAGCCGCCTGACCACACAGAGGCACCTCACTGTGGGAGAAGAGTGGTCTCAGGACCTTCACAGTAGCGGCCGCACAGACCTCCGCTACTCTTACCGGTTTGTGTGTGATGAACACTACTATGGAGAAGGCTGCTCCGTGTTCTGCCGACCGCGGGATGATGCCTTTGGCCACTTCACCTGCGGGGAGAGAGGGGAGAAGATGTGCGACCCTGGCTGGAAAGGCCAGTACTGCACTGACCCCATTTGTCTGCCAGGCTGTGATGACCAACATGGATATTGTGACAAACCGGGGGAATGCAAGTGCAGAGTTGGCTGGCAGGGCCGCTACTGCGATGAATGCATCCGATACCCAGGCTGTCTCCATGGTACCTGCCAGCAGCCCTGGCAGTGTAACTGCCAGGAAGGCTGGGGGGGCCTCTTCTGCAACCAGGATCTGAACTACTGCACTCACCATAAGCCATGCAGGAACGGAGCCACCTGCACCAACACGGGCCAGGGGAGCTACACATGCTCTTGCCGACCCGGGTATACAGGGGCCAACTGTGAGCTGGAGGTAGATGAGTGTGCTCCCAGCCCCTGCAGGAATGGAGGGAGCTGCACGGATCTTGAGGACAGCTACTCTTGCACCTGCCCTCCTGGCTTCTATGGCAAGGTCTGTGAGCTGAGCGCCATGACGTGTGCAGATGGTCCTTGCTTCAATGGGGGACGATGTTCGGATAACCCCGATGGAGGCTACACCTGCCATTGCCCTGCGGGCTTCTCTGGCTTCAACTGTGAGAAGAAGATTGATCTCTGTAGCTCTTCCCCTTGTTCTAACGGTGCCAAGTGTGTGGACCTCGGCAACTCCTACCTGTGCCGATGTCAGACTGGCTTCTCCGGGAGGTACTGCGAGGACAATGTGGATGACTGTGCCTCTTCTCCCTGTGCAAACGGGGGCACCTGCCGGGACAGTGTGAACGATTTCTCCTGTACCTGCCCACCTGGCTACACAGGCAGGAACTGCAGCGCCCCTGTCAGCAGGTGTGAGCATGCACCCTGTCATAACGGGGCCACCTGCCACCAGAGGGGCCAACGCTACATGTGTGAGTGCGCCCAGGGCTATGGCGGCGCCAACTGCCAGTTCCTGCTCCCTGAGCCACCACCAGACCTCATAGTGGCGGCCCAGGGCGGGTCCTTCCCCTGGAGCAGGGCTGACATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGACGGAGGACGTGGTGTGCTGCCACTCAATCTACGGCAAGAAGAAGGGTGATATCGACACCTACCGATACATAGGCTCTTCCGGGACA

TCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTTCTCTACCAGGAGTTCGATGAGATGGAAGAGTGCTCTCAGCACTATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCA

GTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTCT

SEQ ID NO: 66 is a nucleotide sequence encoding hN1.

(SEQ ID NO: 66) ATGCCGCCGCTCCTGGCGCCCCTGCTCTGCCTGGCGCTGCTGCCCGCGCTCGCCGCACGAGGCCCGCGATGCTCCCAGCCCGGTGAGACCTGCCTGAATGGCGGGAAGTGTGAAGCGGCCAATGGCACGGAGGCCTGCGTCTGTGGCGGGGCCTTCGTGGGCCCGCGATGCCAGGACCCCAACCCGTGCCTCAGCACCCCCTGCAAGAACGCCGGGACATGCCACGTGGTGGACCGCAGAGGCGTGGCAGACTATGCCTGCAGCTGTGCCCTGGGCTTCTCTGGGCCCCTCTGCCTGACACCCCTGGACAATGCCTGCCTCACCAACCCCTGCCGCAACGGGGGCACCTGCGACCTGCTCACGCTGACGGAGTACAAGTGCCGCTGCCCGCCCGGCTGGTCAGGGAAATCGTGCCAGCAGGCTGACCCGTGCGCCTCCAACCCCTGCGCCAACGGTGGCCAGTGCCTGCCCTTCGAGGCCTCCTACATCTGCCACTGCCCACCCAGCTTCCATGGCCCCACCTGCCGGCAGGATGTCAACGAGTGTGGCCAGAAGCCCGGGCTTTGCCGCCACGGAGGCACCTGCCACAACGAGGTCGGCTCCTACCGCTGCGTCTGCCGCGCCACCCACACTGGCCCCAACTGCGAGCGGCCCTACGTGCCCTGCAGCCCCTCGCCCTGCCAGAACGGGGGCACCTGCCGCCCCACGGGCGACGTCACCCACGAGTGTGCCTGCCTGCCAGGCTTCACCGGCCAGAACTGTGAGGAAAATATCGACGATTGTCCAGGAAACAACTGCAAGAACGGGGGTGCCTGTGTGGACGGCGTGAACACCTACAACTGCCGCTGCCCGCCAGAGTGGACAGGTCAGTACTGTACCGAGGATGTGGACGAGTGCCAGCTGATGCCAAATGCCTGCCAGAACGGCGGGACCTGCCACAACACCCACGGTGGCTACAACTGCGTGTGTGTCAACGGCTGGACTGGTGAGGACTGCAGCGAGAACATTGATGACTGTGCCAGCGCCGCCTGCTTCCACGGCGCCACCTGCCATGACCGTGTGGCCTCCTTCTACTGCGAGTGTCCCCATGGCCGCACAGGTCTGCTGTGCCACCTCAACGACGCATGCATCAGCAACCCCTGTAACGAGGGCTCCAACTGCGACACCAACCCTGTCAATGGCAAGGCCATCTGCACCTGCCCCTCGGGGTACACGGGCCCGGCCTGCAGCCAGGACGTGGATGAGTGCTCGCTGGGTGCCAACCCCTGCGAGCATGCGGGCAAGTGCATCAACACGCTGGGCTCCTTCGAGTGCCAGTGTCTGCAGGGCTACACGGGCCCCCGATGCGAGATCGACGTCAACGAGTGCGTCTCGAACCCGTGCCAGAACGACGCCACCTGCCTGGACCAGATTGGGGAGTTCCAGTGCATCTGCATGCCCGGCTACGAGGGTGTGCACTGCGAGGTCAACACAGACGAGTGTGCCAGCAGCCCCTGCCTGCACAATGGCCGCTGCCTGGACAAGATCAATGAGTTCCAGTGCGAGTGCCCCACGGGCTTCACTGGGCATCTGTGCCAGTACGATGTGGACGAGTGTGCCAGCACCCCCTGCAAGAATGGTGCCAAGTGCCTGGACGGACCCAACACTTACACCTGTGTGTGCACGGAAGGGTACACGGGGACGCACTGCGAGGTGGACATCGATGAGTGCGACCCCGACCCCTGCCACTACGGCTCCTGCAAGGACGGCGTCGCCACCTTCACCTGCCTCTGCCGCCCAGGCTACACGGGCCACCACTGCGAGACCAACATCAACGAGTGCTCCAGCCAGCCCTGCCGCCACGGGGGCACCTGCCAGGACCGCGACAACGCCTACCTCTGCTTCTGCCTGAAGGGGACCACAGGACCCAACTGCGAGATCAACCTGGATGACTGTGCCAGCAGCCCCTGCGACTCGGGCACCTGTCTGGACAAGATCGATGGCTACGAGTGTGCCTGTGAGCCGGGCTACACAGGGAGCATGTGTAACATCAACATCGATGAGTGTGCGGGCAACCCCTGCCACAACGGGGGCACCTGCGAGGACGGCATCAATGGCTTCACCTGCCGCTGCCCCGAGGGCTACCACGACCCCACCTGCCTGTCTGAGGTCAATGAGTGCAACAGCAACCCCTGCGTCCACGGGGCCTGCCGGGACAGCCTCAACGGGTACAAGTGCGACTGTGACCCTGGGTGGAGTGGGACCAACTGTGACATCAACAACAATGAGTGTGAATCCAACCCTTGTGTCAACGGCGGCACCTGCAAAGACATGACCAGTGGCTACGTGTGCACCTGCCGGGAGGGCTTCAGCGGTCCCAACTGCCAGACCAACATCAACGAGTGTGCGTCCAACCCATGTCTGAACCAGGGCACGTGTATTGACGACGTTGCCGGGTACAAGTGCAACTGCCTGCTGCCCTACACAGGTGCCACGTGTGAGGTGGTGCTGGCCCCGTGTGCCCCCAGCCCCTGCAGAAACGGCGGGGAGTGCAGGCAATCCGAGGACTATGAGAGCTTCTCCTGTGTCTGCCCCACGGGCTGGCAAGCAGGGCAGACCTGTGAGGTCGACATCAACGAGTGCGTTCTGAGCCCGTGCCGGCACGGCGCATCCTGCCAGAACACCCACGGCGGCTACCGCTGCCACTGCCAGGCCGGCTACAGTGGGCGCAACTGCGAGACCGACATCGACGACTGCCGGCCCAACCCGTGTCACAACGGGGGCTCCTGCACAGACGGCATCAACACGGCCTTCTGCGACTGCCTGCCCGGCTTCCGGGGCACTTTCTGTGAGGAGGACATCAACGAGTGTGCCAGTGACCCCTGCCGCAACGGGGCCAACTGCACGGACTGCGTGGACAGCTACACGTGCACCTGCCCCGCAGGCTTCAGCGGGATCCACTGTGAGAACAACACGCCTGACTGCACAGAGAGCTCCTGCTTCAACGGTGGCACCTGCGTGGACGGCATCAACTCGTTCACCTGCCTGTGTCCACCCGGCTTCACGGGCAGCTACTGCCAGCACGATGTCAATGAGTGCGACTCACAGCCCTGCCTGCATGGCGGCACCTGTCAGGACGGCTGCGGCTCCTACAGGTGCACCTGCCCCCAGGGCTACACTGGCCCCAACTGCCAGAACCTTGTGCACTGGTGTGACTCCTCGCCCTGCAAGAACGGCGGCAAATGCTGGCAGACCCACACCCAGTACCGCTGCGAGTGCCCCAGCGGCTGGACCGGCCTTTACTGCGACGTGCCCAGCGTGTCCTGTGAGGTGGCTGCGCAGCGACAAGGTGTTGACGTTGCCCGCCTGTGCCAGCATGGAGGGCTCTGTGTGGACGCGGGCAACACGCACCACTGCCGCTGCCAGGCGGGCTACACAGGCAGCTACTGTGAGGACCTGGTGGACGAGTGCTCACCCAGCCCCTGCCAGAACGGGGCCACCTGCACGGACTACCTGGGCGGCTACTCCTGCAAGTGCGTGGCCGGCTACCACGGGGTGAACTGCTCTGAGGAGATCGACGAGTGCCTCTCCCACCCCTGCCAGAACGGGGGCACCTGCCTCGACCTCCCCAACACCTACAAGTGCTCCTGCCCACGGGGCACTCAGGGTGTGCACTGTGAGATCAACGTGGACGACTGCAATCCCCCCGTTGACCCCGTGTCCCGGAGCCCCAAGTGCTTTAACAACGGCACCTGCGTGGACCAGGTGGGCGGCTACAGCTGCACCTGCCCGCCGGGCTTCGTGGGTGAGCGCTGTGAGGGGGATGTCAACGAGTGCCTGTCCAATCCCTGCGACGCCCGTGGCACCCAGAACTGCGTGCAGCGCGTCAATGACTTCCACTGCGAGTGCCGTGCTGGTCACACCGGGCGCCGCTGCGAGTCCGTCATCAATGGCTGCAAAGGCAAGCCCTGCAAGAATGGGGGCACCTGCGCCGTGGCCTCCAACACCGCCCGCGGGTTCATCTGCAAGTGCCCTGCGGGCTTCGAGGGCGCCACGTGTGAGAATGACGCTCGTACCTGCGGCAGCCTGCGCTGCCTCAACGGCGGCACATGCATCTCCGGCCCGCGCAGCCCCACCTGCCTGTGCCTGGGCCCCTTCACGGGCCCCGAATGCCAGTTCCCGGCCAGCAGCCCCTGCCTGGGCGGCAACCCCTGCTACAACCAGGGGACCTGTGAGCCCACATCCGAGAGCCCCTTCTACCGTTGCCTGTGCCCCGCCAAATTCAACGGGCTCTTGTGCCACATCCTGGACTACAGCTTCGGGGGTGGGGCCGGGCGCGACATCCCCCCGCCGCTGATCGAGGAGGCGTGCGAGCTGCCCGAGTGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTGCAGTGCAACAACCACGCGTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCAATGACCCCTGGAAGAACTGCACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGTGACGGCCACTGTGACAGCCAGTGCAACTCAGCCGGCTGCCTCTTCGACGGCTTTGACTGCCAGCGTGCGGAAGGCCAGTGCAACCCCCTGTACGACCAGTACTGCAAGGACCACTTCAGCGACGGGCACTGCGACCAGGGCTGCAACAGCGCGGAGTGCGAGTGGGACGGGCTGGACTGTGCGGAGCATGTACCCGAGAGGCTGGCGGCCGGCACGCTGGTGGTGGTGGTGCTGATGCCGCCGGAGCAGCTGCGCAACAGCTCCTTCCACTTCCTGCGGGAGCTCAGCCGCGTGCTGCACACCAACGTGGTCTTCAAGCGTGACGCACACGGCCAGCAGATGATCTTCCCCTACTACGGCCGCGAGGAGGAGCTGCGCAAGCACCCCATCAAGCGTGCCGCCGAGGGCTGGGCCGCACCTGACGCCCTGCTGGGCCAGGTGAAGGCCTCGCTGCTCCCTGGTGGCAGCGAGGGTGGGCGGCGGCGGAGGGAGCTGGACCCCATGGACGTCCGCGGCTCCATCGTCTACCTGGAGATTGACAACCGGCAGTGTGTGCAGGCCTCCTCGCAGTGCTTCCAGAGTGCCACCGACGTGGCCGCATTCCTGGGAGCGCTCGCCTCGCTGGGCAGCCTCAACATCCCCTACAAGATCGAGGCCGTGCAGAGTGAGACCGTGGAGCCGCCCCCGCCGGCGCAGCTGCACTTCATGTACGTGGCGGCGGCCGCCTTTGTGCTTCTGTTCTTCGTGGGCTGCGGGGTGCTGCTGTCCCGCAAGCGCCGGCGGCAGCATGGCCAGCTCTGGTTCCCTGAGGGCTTCAAAGTGTCTGAGGCCAGCAAGAAGAAGCGGCGGGAGCCCCTCGGCGAGGACTCCGTGGGCCTCAAGCCCCTGAAGAACGCTTCAGACGGTGCCCTCATGGACGACAACCAGAATGAGTGGGGGGACGAGGACCTGGAGACCAAGAAGTTCCGGTTCGAGGAGCCCGTGGTTCTGCCTGACCTGGACGACCAGACAGACCACCGGCAGTGGACTCAGCAGCACCTGGATGCCGCTGACCTGCGCATGTCTGCCATGGCCCCCACACCGCCCCAGGGTGAGGTTGACGCCGACTGCATGGACGTCAATGTCCGCGGGCCTGATGGCTTCACCCCGCTCATGATCGCCTCCTGCAGCGGGGGCGGCCTGGAGACGGGCAACAGCGAGGAAGAGGAGGACGCGCCGGCCGTCATCTCCGACTTCATCTACCAGGGCGCCAGCCTGCACAACCAGACAGACCGCACGGGCGAGACCGCCTTGCACCTGGCCGCCCGCTACTCACGCTCTGATGCCGCCAAGCGCCTGCTGGAGGCCAGCGCAGATGCCAACATCCAGGACAACATGGGCCGCACCCCGCTGCATGCGGCTGTGTCTGCCGACGCACAAGGTGTCTTCCAGATCCTGATCCGGAACCGAGCCACAGACCTGGATGCCCGCATGCATGATGGCACGACGCCACTGATCCTGGCTGCCCGCCTGGCCGTGGAGGGCATGCTGGAGGACCTCATCAACTCACACGCCGACGTCAACGCCGTAGATGACCTGGGCAAGTCCGCCCTGCACTGGGCCGCCGCCGTGAACAATGTGGATGCCGCAGTTGTGCTCCTGAAGAACGGGGCTAACAAAGATATGCAGAACAACAGGGAGGAGACACCCCTGTTTCTGGCCGCCCGGGAGGGCAGCTACGAGACCGCCAAGGTGCTGCTGGACCACTTTGCCAACCGGGACATCACGGATCATATGGACCGCCTGCCGCGCGACATCGCACAGGAGCGCATGCATCACGACATCGTGAGGCTGCTGGACGAGTACAACCTGGTGCGCAGCCCGCAGCTGCACGGAGCCCCGCTGGGGGGCACGCCCACCCTGTCGCCCCCGCTCTGCTCGCCCAACGGCTACCTGGGCAGCCTCAAGCCCGGCGTGCAGGGCAAGAAGGTCCGCAAGCCCAGCAGCAAAGGCCTGGCCTGTGGAAGCAAGGAGGCCAAGGACCTCAAGGCACGGAGGAAGAAGTCCCAGGACGGCAAGGGCTGCCTGCTGGACAGCTCCGGCATGCTCTCGCCCGTGGACTCCCTGGAGTCACCCCATGGCTACCTGTCAGACGTGGCCTCGCCGCCACTGCTGCCCTCCCCGTTCCAGCAGTCTCCGTCCGTGCCCCTCAACCACCTGCCTGGGATGCCCGACACCCACCTGGGCATCGGGCACCTGAACGTGGCGGCCAAGCCCGAGATGGCGGCGCTGGGTGGGGGCGGCCGGCTGGCCTTTGAGACTGGCCCACCTCGTCTCTCCCACCTGCCTGTGGCCTCTGGCACCAGCACCGTCCTGGGCTCCAGCAGCGGAGGGGCCCTGAATTTCACTGTGGGCGGGTCCACCAGTTTGAATGGTCAATGCGAGTGGCTGTCCCGGCTGCAGAGCGGCATGGTGCCGAACCAATACAACCCTCTGCGGGGGAGTGTGGCACCAGGCCCCCTGAGCACACAGGCCCCCTCCCTGCAGCATGGCATGGTAGGCCCGCTGCACAGTAGCCTTGCTGCCAGCGCCCTGTCCCAGATGATGAGCTACCAGGGCCTGCCCAGCACCCGGCTGGCCACCCAGCCTCACCTGGTGCAGACCCAGCAGGTGCAGCCACAAAACTTACAGATGCAGCAGCAGAACCTGCAGCCAGCAAACATCCAGCAGCAGCAAAGCCTGCAGCCGCCACCACCACCACCACAGCCGCACCTTGGCGTGAGCTCAGCAGCCAGCGGCCACCTGGGCCGGAGCTTCCTGAGTGGAGAGCCGAGCCAGGCAGACGTGCAGCCACTGGGCCCCAGCAGCCTGGCGGTGCACACTATTCTGCCCCAGGAGAGCCCCGCCCTGCCCACGTCGCTGCCATCCTCGCTGGTCCCACCCGTGACCGCAGCCCAGTTCCTGACGCCCCCCTCGCAGCACAGCTACTCCTCGCCTGTGGACAACACCCCCAGCCACCAGCTACAGGTGCCTGAGCACCCCTTCCTCACCCCGTCCCCTGAGTCCCCTGACCAGTGGTCCAGCTCGTCCCCGCATTCCAACGTCTCCGACTGGTCCGAGGGCGTCTCCAGCCCTCCCACCAGCATGCAGTCCCAGATCGCCCGCATTCCGGAGGCCTTCAAGGCTAGCTAA.

SEQ ID NO: 67 is a nucleotide sequence encodingmyc-moxGFP-mN1TMD-GAL4-VP64.

(SEQ ID NO: 67) GCCACCATGGCATTGCCCGTGACCGCCCTGCTGCTGCCACTGGCCTTGTTGCTCCACGCCGCGCGGCCAGAACAGAAGCTGATCAGCGAGGAGGATCTGACCGGTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCTCCGTGCGGGGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCAGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGAGCTTCTCCCGCTACCCCGACCACATGAAGCGCCACGACTTCTTCAAGAGCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCCTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACTCCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACGTGGAGGACGGCTCCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGTCCACCCAGTCCAAGCTGTCCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTTCTGGAATTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGGGATCCCCGGTGGAGCCTCCGCTGCCCTCGCAGCTGCACCTCATGTACGTGGCAGCGGCCGCCTTCGTGCTCCTGTTCTTTGTGGGCTGTGGGGTGCTGCTGTCCCGCAAGCGCCGGCGGGGCTCGAGCATGAAGCTGCTGAGCAGCATCGAGCAGGCCTGTGACATCTGCCGGCTGAAGAAACTGAAGTGCAGCAAAGAAAAGCCCAAGTGCGCCAAGTGCCTGAAGAACAACTGGGAGTGCCGGTACAGCCCCAAGACCAAGAGAAGCCCCCTGACCAGAGCCCACCTGACCGAGGTGGAAAGCCGGCTGGAAAGACTGGAACAGCTGTTTCTGCTGATCTTCCCACGCGAGGACCTGGACATGATCCTGAAGATGGACAGCCTGCAGGACATCAAGGCCCTGCTGACCGGCCTGTTCGTGCAGGACAACGTGAACAAGGACGCCGTGACCGACAGACTGGCCAGCGTGGAAACCGACATGCCCCTGACCCTGCGGCAGCACAGAATCAGCGCCACCAGCAGCAGCGAGGAAAGCAGCAACAAGGGCCAGCGGCAGCTGACAGTGTCTGCTGCTGCAGGCGGAAGCGGAGGCTCTGGCGGATCTGATGCCCTGGACGACTTCGACCTGGATATGCTGGGCAGCGACGCCCTGGATGATTTTGATCTGGACATGCTGGGATCTGACGCTCTGGACGATTTCGATCTCGACATGTTGGGATCAGATGCACTGGATGACTTTGACCTGGACATGCTCGGATCATGA

SEQ ID NO: 68 is an amino acid sequence encodingmyc-moxGFP-mN1TMD-GAL4-VP64Bolded text indicates the CD8alpha signalsequence; Bolded, underlined text indicates the myc epitope; Italicizedtext indicates moxGFP (e.g., as described in Costantini, et al. 2015);Double underlined text indicates the mouse Notch1 juxtamembrane andtransmembrane domains; and

indicates GAL4-VP64 Activator.

(SEQ ID NO: 68) MALPVTALLLPLALLLHAARP EQKLISEEDLTGVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDEVKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRRNVEDGSVQLADEYQQNTPIGDGPVILPDNHYLSTQSKLSEDPNEKRDEMVILEFVTAA

1. A synthetic inhibitor or auto-inhibitory protein comprising a NotchNRR (Negative Regulatory Region)-binding scFV fused to a transmembranedomain.
 2. The synthetic protein of claim 1, wherein the Notch NRRcomprises a Notch NRR1 of SEQ ID NO:
 8. 3. The synthetic protein ofclaim 1, wherein the Notch NRR is mutated relative to Notch NRR1 of SEQID NO:
 8. 4. The synthetic protein of claim 1, further comprising asynthetic Notch receptor protein comprising a mutated Notch NRR.
 5. Anengineered cell comprising the synthetic inhibitor or auto-inhibitoryprotein of claim
 1. 6. The engineered cell of claim 5, wherein theengineered cell is an engineered T cell.
 7. A synthetic Notch receptorprotein comprising, from N-terminal to C-terminal and in covalentlinkage, (i) a ligand binding domain (LBD), (ii) a mutated Notch NRR(Negative Regulatory Region), (iii) a transmembrane domain, and (iv) anintracellular domain, wherein the mutated Notch NRR is bound withhigh-affinity by a synthetic inhibitor protein comprising a mutatedNotch NRR-binding scFV fused to a transmembrane domain.
 8. The syntheticprotein of claim 7, wherein the mutated Notch NRR is mutated relative toNotch NRR1 of SEQ ID NO:
 8. 9. A synthetic Notch receptor proteincomprising, from N-terminal to C-terminal and in covalent linkage, (i) aligand binding domain (LBD), (ii) a scFV that binds to an at least oneNotch NRR (Negative Regulatory Region), (iii) a Notch NRR bound by thescFV, (iv) a transmembrane domain, and (v) an intracellular domain. 10.The synthetic protein of claim 9, wherein the Notch NRR comprises aNotch NRR1 of SEQ ID NO:
 8. 11. The synthetic protein of claim 9,wherein the Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8.12. A synthetic, drug-dependent protein comprising a ligand bindingdomain (LBD), an NS3 protease domain, and a transmembrane domain, andwherein the NS3 is located in between the protease domain.
 13. Thesynthetic, drug-dependent protein of claim 12, wherein the LBD andtransmembrane domain comprise a sequence of human Delta ligand.
 14. Thesynthetic, drug-dependent protein of claim 12, wherein the NS3 domaincomprises a sequence of SEQ ID NO:
 32. 15. The synthetic, drug-dependentprotein of claim 12, further comprising a targeting domain.
 16. Thesynthetic protein of claim 1, wherein the transmembrane domain comprisesthe human Notch1 transmembrane domain of SEQ ID NO:
 13. 17. Thesynthetic protein of claim 7, wherein the transmembrane domain comprisesthe human Notch1 transmembrane domain of SEQ ID NO:
 13. 18. Thesynthetic protein of claim 9, wherein the transmembrane domain comprisesthe human Notch1 transmembrane domain of SEQ ID NO:
 13. 19. Thesynthetic protein of claim 12, wherein the transmembrane domaincomprises the human Notch1 transmembrane domain of SEQ ID NO:
 13. 20.The synthetic protein of claim 1, wherein the scFV comprises, fromN-terminal to C-terminal and in covalent linkage, a V_(H) domain, alinker domain, and a V_(L) domain.
 21. The synthetic protein of claim 7,wherein the scFV comprises, from N-terminal to C-terminal and incovalent linkage, a V_(H) domain, a linker domain, and a V_(L) domain.22. The synthetic protein of claim 9, wherein the scFV comprises, fromN-terminal to C-terminal and in covalent linkage, a V_(H) domain, alinker domain, and a V_(L) domain.
 23. The synthetic protein of claim 1,wherein the scFV is selected from any one of SEQ ID NOs: 15-27.
 24. Thesynthetic protein of claim 7, wherein the scFV is selected from any oneof SEQ ID NOs: 15-27.
 25. The synthetic protein of claim 9, wherein thescFV is selected from any one of SEQ ID NOs: 15-27.
 26. The syntheticprotein of claim 1, further comprising a signal sequence N-terminal tothe LBD.
 27. The synthetic protein of claim 7, further comprising asignal sequence N-terminal to the LBD.
 28. The synthetic protein ofclaim 9, further comprising a signal sequence N-terminal to the LBD.